scholarly journals Complement Activation during Vaso-Occlusive Pain Crisis in Pediatric Sickle Cell Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 858-858
Author(s):  
Justin J Yoo ◽  
Sara H Graciaa ◽  
Jayre A Jones ◽  
Zoey Zuo ◽  
Connie M Arthur ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Complement Activation ◽  
Research Funding ◽  
Cell Disease ◽  
Intravascular Hemolysis ◽  
Acute Complications ◽  
Steady State Levels ◽  
Pain Crisis

Abstract Background: Sickle cell disease (SCD) affects millions of individuals worldwide with substantial morbidity and mortality. The sickle hemoglobin (HbS) polymerizes upon deoxygenation, causing rigid and adhesive red blood cells (RBCs), triggering vascular occlusion, greatly shortened RBC lifespan, and chronic hemolysis. Amongst acute complications in SCD, vaso-occlusive pain crisis (VOC) is the leading cause of hospitalization, with supportive care being the primary approach to management. We and others have recently demonstrated important contributions of complement to the pathophysiology of SCD. When the complement pathway (CP) is activated during SCD crises, inhibition at C5 using eculizumab, has been successful in treating various acute complications in SCD (Chonat et al, Haematologica). In this study, we prospectively analyzed the extent of CP activation among children with SCD presenting with VOC. Methods: Patients aged 0-21 years old managed at Children's Healthcare of Atlanta with homozygous sickle cell (SS) or S beta zero thalassemia genotypes were enrolled in an IRB-approved research study. Inclusion criteria included those requiring intravenous opioids for VOC, and excluded those with chronic pain, >6 VOC admission in the previous 12 months or on chronic transfusions. Blood samples were collected within 48 hours of VOC presentation, and steady-state levels were obtained at a 4-week clinic follow-up. Data was analyzed using a paired t-test, and receiver operator characteristic (ROC) curves were generated comparing intra-person complement levels during acute VOC versus respective steady-state levels. Results: Sixty-four patients have been enrolled thus far, of which 43 (67%) had steady-state samples collected. The majority of patients (90.5%) have SS genotype with a mean (SD) patient age of 14.15 (4.68) years. Fifty-three (84.1%) patients reported taking hydroxyurea (HU). Fifty-nine (93.7%) patients had at least one VOC admission in the past 12 months, with an average of 2.98 (1.67) VOC admissions. Pain Score reported on 55 patients averaged 4.93 (4.78) on a pain scale of 0 to 10. Mean values during VOC and steady-state of hemoglobin (Hb) were 8.12 and 9.01 g/dL, platelet count 431 and 511, and lactate dehydrogenase (LDH) 549 and 483 U/L, respectively. Seventeen patients had complement work-up performed during acute and steady-state, and 4 of them had additional samples collected during subsequent VOC. Complement protein levels C3, C4, C5, properdin, factor B, and complement regulatory proteins factor H and I were unremarkable during VOC and steady-state. However, complement activation markers, specifically anaphylatoxins C3a, C5a and Bb were significantly elevated during VOC compared to steady-state (see Table 1) suggesting activation of alternative CP during VOC (see Table 1 and Figure 1A-C). Terminal complement complex (C5b9) was not statistically different between VOC and steady-state (Figure 1D, red dotted lines signify normal ranges). Remarkably, patients who re-presented with acute VOC exhibited similar increases in their C3a/C5a (Figure 1E-F), substantiating the increases related to their VOC. Hemoglobin and LDH (Figure 1G-H) were similarly significant, suggestive of intravascular hemolysis. Three (7.1%) patients developed acute chest syndrome, two of whom experienced respiratory failure requiring intensive care management, and all exhibited significant CP activation. The area under the curve (AUC) of the ROC curve was analyzed to determine the ability of complement biomarkers to differentiate VOC from steady-state. Based on the AUC of these biomarkers, complement anaphylatoxins C3a and C5a exhibited the highest AUC of 0.76 and 0.87, respectively. Discussion: To our knowledge, this is the first prospective and comprehensive evaluation of CP in patients with SCD during VOC and steady-states. These preliminary findings suggest CP activation is present in a large proportion of patients during VOC, with increased activation of alternative and common CP, associated with intravascular hemolysis. Minimal increase in C5b9 could be explained by a significant proportion (> 80%) of our patients being on HU therapy, similar to prior data (Roumenina et al, AJH). Specifically, C3a/C5a, along with other biomarkers, could not only predict disease activity in patients during VOC, but provide pharmacological targets in VOC, which need further validation. Figure 1 Figure 1. Disclosures Stowell: Alexion: Consultancy; Argenx: Speakers Bureau; Grifols: Speakers Bureau. Chonat: Alexion: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Takeda: Consultancy, Research Funding.

Complement Activation in a Murine Model of Sickle Cell Disease: Inhibition of Vaso-Occlusion By Blocking C5 Activation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 158-158 ◽  
Author(s):  
Terry Robert Schaid ◽  
Julia Nguyen ◽  
Chunsheng Chen ◽  
Fuad Abdulla ◽  
Trevor Killeen ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Complement Activation ◽  
Research Funding ◽  
Annexin V ◽  
Dorsal Skin ◽  
Cell Disease ◽  
Activation Markers ◽  
Isotype Control ◽  
Skin Fold

Abstract Introduction Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. The alternative complement pathway is abnormally activated in SCD and is additionally activated by phosphatidylserine (PS) on the outer leaflet of SS-red blood cells (SS-RBC). PS on the surface of SS-RBC and activated platelets accelerates the assembly of prothrombinase complexes leading to generation of thrombin, which can cleave circulating C5 protein into two biologically active fragments, C5a and C5b. C5a is an anaphylatoxin, a potent pro-inflammatory mediator, that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion (VO). Active C5b fragments stimulate the formation of membrane attack complexes (MAC) on SS-RBC that increases their susceptibility to lysis. We hypothesize that complement activation on the surface of SS-RBC may stimulate VO and RBC turnover in SCD. Methods and Results Whole blood from Townes-AA, -AS, and -SS mice (n=4) was collected in EDTA and RBC were immunostained with erythroid specific anti-Ter119 (Ly-76) IgG conjugated to PE-Cy7, anti-C5b-9 (MAC) IgG conjugated to Alexa Fluor 647, and anti-C3 (also specific for activation fragments C3b, iC3b, C3d and C3dg) IgG conjugated to PE. The percentages of Ter119-positive RBC that were positive for MAC were 6.3%, 6.5%, and 26.0% for AA-, AS-, and SS-RBCs, respectively (p<0.01 SS vs. AA and AS) suggesting enhanced C5 activation in Townes-SS mice. However, EDTA plasma C5a levels measured by ELISA were not significantly different between Townes-AA, -AS and -SS mice. C3 activation fragments were also found on a subset of MAC positive RBC. We used a mouse model of hypoxia/reoxygenation (H/R)-induced stasis to investigate the role of complement in VO. Townes-SS mice with implanted dorsal skin-fold chambers were infused with 30 µg of either anti-C5 IgG mAb BB5.1 (which blocks murine C5 cleavage ) (n=4), annexin V (n=2), or with isotype control IgG (n=4) 30 minutes prior to H/R (1 hour of hypoxia at 7% O2, followed by reoxygenation for 4 hours in room air). Percent microvascular stasis (% non-flowing venules) in the subcutaneous venules in the dorsal skin fold chamber window was measured using intravital microscopy at 1 and 4 hours post-hypoxia. Percent stasis was significantly lower in the Townes-SS mice receiving anti-C5 IgG or annexin V than the mice receiving the isotype control IgG at 1 and 4 hours post-hypoxia (Figure 1, means ± SD, *p<0.01 anti-C5 IgG or annexin V vs. control IgG). This finding implies that H/R may acutely activate C5 , thus leading to stasis. Initial studies suggest increased plasma C5a levels in Towne-SS mice treated with isotype control IgG compared to mice treated with anti-C5 IgG in response to H/R. Conclusions These results demonstrate an increased percentage of SS-RBC expressing MAC on their surface and inhibition of H/R-induced stasis with anti-C5 IgG. However, circulating C5a levels were not different between untreated Townes-AA, -AS, or -SS mice suggesting that C5a is not chronically elevated in SCD. Rather, the generation of C5a may occur locally and in the acute setting. Current studies underway are examining the effects of anti-C5 IgG on MAC deposition and RBC half-lives; the mechanism(s) of complement activation in SCD mice (classical, alternative, tic-over, etc); the ability of recombinant murine C5a to induce stasis; and complement activation markers on RBC and in plasma from SCD patients and controls. Disclosures Chen: Imara: Research Funding. Belcher:Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:Imara: Research Funding; CSL-Behring: Research Funding.

scholarly journals High-Throughput Mirna Analysis Suggests Pro-Inflammatory Profile in Sickle Cell Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1077-1077
Author(s):  
Matthew Cannon ◽  
Sarah Glass ◽  
Sidney Smith ◽  
Melanie Heinlein ◽  
Rosa Lapalombella ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Research Funding ◽  
Red Cell ◽  
Cell Disease ◽  
Acute Crisis

Abstract BACKGROUND: Mature circulating red blood cells, though devoid of a nucleus, have been shown to contain an abundance of miRNAs. Further, it has been shown that sickle cell patient-derived RBCs have a dramatic difference in miRNA content than normal RBCs. Given that a range of miRNAs are involved in the regulation of immunity, including the release of inflammatory mediators, we hypothesize that miRNAs enriched in circulating red blood cells function to prolong the inflammatory state in sickle cell disease. Further, we hypothesize that these miRNAs can be used as biomarkers for use in the clinic to predict crisis and differentiate acute versus chronic pain. Exploring this miRNA enrichment in circulating red blood cells in sickle cell patients will provide practical insight for the inflammation state and will inform characteristics of patients who may need greater care in the clinic. METHODS: Twenty steady state patients were recruited and categorized according to their chronic pain status and crisis frequency per year. Whole blood was drawn during routine visits to the OSU Wexner Medical Center Hematology Clinic. Additionally, whole blood was drawn from five patients either in acute pain crisis (recruited prior to crisis) or within a few days of crisis. Samples were subject to double gradient centrifugation and red cells were resuspended in Trizol and cryopreserved. MiRNAs were isolated from red cell Trizol suspensions using a commercial isolation kit (QIAGEN Cat#217004). Isolated miRNAs were then subject to a NanoString Human miR (v3) expression assay. Differential expression analysis was conducted to compare miRNAs with at least 1.5 fold difference (p = 0.05) between steady state and acute crisis. Target prediction and GO ontology analysis was performed for statistically significant miRNAs using DIANA Tools mirPath v3. Follow-up qPCRs were performed using TaqMan Advanced miRNA cDNA Synthesis Kit (Cat#A28007) and TaqMan Advanced miRNA Assays (Cat#A25576) to validate the decreased expression of miRNAs. Additional qPCRs were performed using TaqMan Gene Expression Assays (Cat#4331182) to investigate mRNA regulatory effects of significant miRNAs in the total red cell population. Western blots were also performed to investigate regulatory effects of these miRNAs at the protein level. RESULTS & CONCLUSION: Comparison of RBC miRNA profiles from patients during acute crisis to those in steady state shows several significantly decreased (>1.5 fold) miRNAs in crisis. Among these miRs we have found previously uncharacterized miRNAs, hsa-miR-2116-5p and hsa-miR-302d-3p. DIANA tools miRNA analysis software predicts these miRNAs to be involved in regulation of cell-to-cell adhesion pathways through gene transcripts such as Protocadherin Beta 6 (PCDHB6) and Neural Cell Adhesion Molecule 2 (NCAM2). Interestingly, inspection of miRNA predicted targets that fall under significant GO terms also predicts several individual miRNAs to regulate inflammatory response and nociceptive signaling gene transcripts like A20 (TNFAIP3) and Cathepsin S (CTSS). Validation of these miRNAs was performed via qPCR for 5 out of the 6 significantly decreased miRNAs. Of the 5 miRNAs tested, hsa-miR-2116-5p, hsa-miR-302d-3p, and hsa-miR-1246 were validated as having decreased expression in acute crisis patients compared to steady state. qPCRs were then performed to probe for miRNA based regulation of top predicted target mRNA transcripts. Both CTSS and TNFAIP3 showed increased expression of mRNA transcripts in acute crisis patient red cells as compared to steady state. Next, western blot analysis was performed on red cell protein lysate. Interestingly, this analysis revealed a pattern in activated CTSS expression that was independent of acute crisis. Steady state patients reporting chronic pain showed increased activated CTSS compared to those without chronic pain. Activated CTSS was not found in red cell lysates from three normal, non-SCD donors. Taken together, these results suggest that red blood cells may play a larger role in inflammation and pain responses in sickle cell disease than previously thought. Further these results suggest activated CTSS as a potential biomarker for differentiating chronic pain in patients. Follow-up studies are underway to further stratify and investigate these findings. Disclosures Desai: University of Pittsburgh: Research Funding; Ironwood: Other: Adjudication Committee; NIH: Research Funding; FDA: Research Funding; Selexy/Novartis: Research Funding; Pfizer: Research Funding.

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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3662-3662
Author(s):  
Mehdi Nouraie ◽  
Debra L Weiner ◽  
Mariana Hildesheim ◽  
Ward Hagar ◽  
Oswaldo L Castro ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Hospital Stay ◽  
Sickle Cell ◽  
Research Funding ◽  
Ischemia Reperfusion ◽  
Serum Lactate Dehydrogenase ◽  
Index Hospitalization ◽  
Cell Disease ◽  
Readmission Risk

Abstract 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 &lt;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. Figure Figure. Table Table. Disclosures Lanzkron: NKT: Research Funding; Selexys: Research Funding; Prolong: Research Funding; PCORI: Research Funding; NHLBI: Research Funding; HRSA: Research Funding; GBT: Consultancy; Pfizer: Research Funding.

scholarly journals The Effect of Individualized Pain Plans for Patients with Sickle Cell Disease on Emergency Provider Satisfaction: A Win for the Patient Can be a Win for the Provider

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1889-1889
Author(s):  
Sherraine Della-Moretta ◽  
Rui Li ◽  
David Way ◽  
Michael G (MD) Purcell ◽  
Melanie Heinlein ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Emergency Room ◽  
Sickle Cell ◽  
Research Funding ◽  
Treatment Decision ◽  
Treatment Decisions ◽  
Cell Disease ◽  
Satisfaction With Treatment ◽  
Pain Crisis ◽  
The Relationship

Abstract Background Sickle cell disease (SCD) is an inherited hematologic disorder that affects approximately 100,000 Americans and results in over 200,000 emergency departments visits annually, largely due to pain (Lanzkron et al). Delay in treatment in emergency room has been a significant barrier to patients with SCD, particularly adults. The objective of this study is to determine the effects of utilizing individualized pain plans for the treatment of vaso-occlusive crisis (VOC) on the satisfaction of healthcare providers in the emergency department (ED). Methods The Ohio State University has a comprehensive sickle cell center which creates individualized pain plans for patients who present to the ED with pain related to VOC. In January 2015, these pain plans were implemented into the electronic medical record listed in the overview of the problem sickle cell disease in each chart. In addition to creating pain plans, an interdisciplinary team was formed consisting of hematologists, pharmacists, and ED providers with the goal of education regarding SCD and the new implementation of pain plans. Surveys, using the secure web application, RedCAP, were distributed to the emergency department providers at the OSU ED. Questions included responders' role in the ED, prior experience with treating pain crisis, time to make treatment management decisions for VOC, satisfaction with treatment decision, and the providers view of their relationship with patients. Wilcoxon signed-rank test and Fisher's exact test were applied to evaluate the differences between pre and post survey numeric and categorical responses. Results Surveys were sent electronically to 170 ED providers. Sixty-nine responses, making up 40.5% of those surveyed, were obtained from 30 attending physicians, 2 fellows, 22 nurse practitioners or physician assistants, 1 registered nurse, and 14 residents. Of those who answered the survey, 14 had experience with treating pain crisis prior to the implementation of individualized pain plans. Implementation of individualized pain plans led to a reduction in median time to make treatment decisions from 5.5 to 2.5 minutes with a p-value of 0.0161. Provider satisfaction with treatment decisions improved as well (p = 0.0029) (Figure 2). In addition, ED providers felt more satisfied with their relationship with patients (p = 0.0078) (Figure 3). The majority of responders (91.2%) also rated their satisfaction with the treatment decision as either satisfied or very satisfied (Figure 1). Seventy eight percent of those answering the survey rated with relationship with patients as being good or very good (Figure 1). In terms of the ease of finding the pain plan in the electronic medical record, 91.3% of providers found them to be either very easy or easy to locate with 94.12% responding that implementing the plan was either easy or very easy (Figure 4). Regarding efficacy of the pain plans, 89.85% found the pain plans to be either effective or very effective (Figure 5). Finally, of the 36 providers who worked elsewhere, about half of the institutions from which they came did not have pain plans. Discussion The results of this study show the importance of utilizing individualized pain plans in the treatment of VOC in the ED. As shown in our prior studies, the implementation of individualized pain plans for patients with SCD resulted in a 48% decrease in time to first opioid in the ED, thereby signifying more prompt treatment (Della-Moretta et al). Not only does the data support an improvement in time to make treatment decisions, which benefit the patients, but providers also appear to view their use as an advantage. Pain plan utilization also leads to an increase in provider confidence in their treatment plans as well as a perceived improvement in patient-provider relationships. This is particularly significant as historically the relationship between emergency room staff and sickle cell patients has been seen as challenging by both patients as well emergency room providers (Haywood et al). Making patient centered individualized pain plans readily available, easily accessible, and simple to enact, can further enhance the relationship between the patient, emergency room, and the hematology team. Ongoing communication and education between all parties is beneficial. With the combination of patient and provider data, we show that a win for the patient can also be a win for the provider. Figure 1 Figure 1. Disclosures Desai: Pfizer: Other: Publication Fee, Research Funding; Novartis: Research Funding, Speakers Bureau; Global Blood Therapeutics: Honoraria, Research Funding; Foundation for Sickle Cell Research: Honoraria; Forma: Consultancy.

scholarly journals The Use of Marijuana and Hydroxyurea Among Sickle Cell Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5913-5913 ◽  
Author(s):  
Nene Kalu ◽  
Patricia A. O'Neal ◽  
Catherine Nwokolo ◽  
Sharmin Diaz ◽  
Oluwakemi Owoyemi
Keyword(s):  
Steady State ◽  
Side Effects ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Severe Pain ◽  
Marijuana Use ◽  
Data Capture ◽  
Cell Disease ◽  
Significant Difference ◽  
Pain Crisis

Abstract Objective Sickle cell crises are a common, painful complication of sickle cell disease. These crises are treated in the emergency department with narcotic analgesia. However due to concerns about addiction, tolerance and side effects of pain medications, physicians specifically hematologists recommend the use of hydroxyurea as an alternative therapeutic medication for sickle cell patients. Hydroxyurea is the only FDA approved therapeutic medication proven to decrease the painful vasoocclusive process in sickle cell disease. Similarly, marijuana is being studied as an alternative in reducing pain in sickle cell disease. However, few have looked at the prevalence of hydroxyurea and marijuana use in sickle cell disease patients. We want to explore the prevalence of marijuana use among our hydroxyurea and non-hydroxyurea users. Method We developed a structured self-administered anonymous questionnaire that was provided to our adult sickle cell patients. One hundred and three (103) patients with HbSS, HbSC, HbSBetaThalassemia and HbSS disease with Alpha Thalassemia trait were consented and explained the purpose of the questionnaire. The questionnaire was administered in a private room in our clinic. The questionnaire focused on the following variables: (1) current regimen of their prescribed narcotic medication at steady state (2) current regimen of their prescribed narcotic medication during a moderate to severe pain crisis (3) use of Marijuana and Hydroxyurea during steady state and during a moderate to severe pain crisis (4) whether or not they were taking Hydroxyurea and the (5) physical effects of combining prescribed narcotic medication with illicit drug use. The questionnaire consisted of twenty-five (25) questions which took on average 10-15 minutes. The answers to the questionnaire were entered into REDCap (Research Electronic Data Capture). It is a secure, web-based application designed to support data capture for research studies, providing 1) an intuitive interface for validated data entry; 2) audit trails for tracking data manipulation and export procedures; 3) automated export procedures for seamless data downloads to common statistical packages; and 4) procedures for importing data from external sources. The data was analyzed using SPSS 23.0 (IBM Corp. Armonk, NY). Chi-square or Fisher exact testing was used to test the association between variables on the prevalence of cannabis use for pain management and other symptom relief and its side effects during self-administration in patients with sickle cell disease. Results Our analysis showed that fifty-one (51%) percent of the participants were females while forty-nine (49%) percent were males. Thirty-six (36%) percent of patients were between 25-34 years of age and the median age was 30 years. Thirty-three percent (33%) had a college degree or higher. Sixty percent (60%) of participants admitted to using marijuana at least once. Fifty-six percent (56%) of patients used both hydroxyurea and marijuana while forty-four percent (44%) of patients did not use hydroxyurea but used marijuana. Thirty percent (30%) of participants stated they used marijuana within the last 12 months to relieve symptoms associated with sickle cell disease. The main reasons for use were to increase appetite, improve sleep, mood and concentration, relieve stress and anxiety and alleviate pain. Eighty percent (80%) of participants stated that marijuana was not as effective in managing their sickle-related pain as hydroxyurea. There was no significant difference (p= 0.173) between the use of marijuana among hydroxyurea users and non-hydroxyurea users. Five percent(5%) of participants reported combining marijuana with prescribed narcotic medications to alleviate their sickle-related pain. Conclusion In summary, our data showed that within the groups of hydroxyurea users and non-hydroxyurea users, there was no significant difference in the use of marijuana. Both groups use marijuana as much as the general population. However, the use of marijuana among our sickle cell patients was not solely dependent on pain control. Further studies need to be conducted to show any significant results between hydroxyurea and non-hydroxyurea users. Disclosures No relevant conflicts of interest to declare.

scholarly journals Severe Acute Complications of Sickle Cell Disease in Two Expert Referral Centers (UK and Italy): Natural History Studies Highlight Ongoing Unmet Need for Effective Disease Modifying or Curative Therapies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3093-3093
Author(s):  
Raffaella Colombatti ◽  
Cynthia Sangarappillai ◽  
Muriel Soriano ◽  
Jonathan Bestwick ◽  
Will Hann ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Board Of Directors ◽  
Sickle Cell ◽  
Real World ◽  
Research Funding ◽  
Standard Of Care ◽  
Cell Disease ◽  
Advisory Committees ◽  
Treatment Groups ◽  
Acute Complications

Abstract Background: Hydroxyurea (HU) and chronic transfusion therapy (CTT) are the only disease modifying therapies (DMTs) currently available as standard of care in the UK and Italy for patients with sickle cell disease (SCD), with hematopoietic stem cell transplant (HSCT) available to a small fraction of patients. Few registries capture long-term follow up of real-world outcomes among patients with SCD and there is a need for natural history studies demonstrating morbidity and mortality under existing standard of care. The East London Newborn Sickle Cohort Study (ELNSCS) at the Royal London Hospital and the Sickle Cell Disease Cohort (SCDC) at the University of Padova (UNIPD) collect biomarker and clinical data on patients followed comprehensively at two expert referral centers and provide an opportunity to understand SCD real-world outcomes. Here, we report severe acute complications under standard of care during years when HU and CTT were widely available and accepted. Methods: Inclusion of patients in the ELNSCS required being born in a designated region in London, diagnosed by newborn screening between 1983-2018 and, for this analysis, followed during 2015-2018. Inclusion in the SCDC required being followed, for this analysis, at the UNIPD during 2016-2019. Analyses were restricted to patients with β Sβ S, β Sβ 0, and, for ELNSCS, β Sβ +. Data were entered into clinical databases at each site and subsequently validated against institutional records. Severe vaso-occlusive events (VOEs) were defined as events requiring admission (inpatient, ED, or hematology day unit) for acute chest syndrome (ACS), acute painful crisis, acute hepatic/splenic sequestration, acute ischaemic stroke, dactylitis and priapism. Statistical analysis was aligned between both sites. Patients were categorized into three mutually exclusive treatment groups (HU, CTT, no treatment) according to treatment during study period. Results: One hundred seventy-two patients in the ELNSCS and 62 patients at UNIPD met study inclusion criteria in the four-year analysis period. HbSS genotype accounted for 161 (93.6%) of ELNSCS cohort and 58 (93.5%) of UNIPD cohort. Median age at study entry was 11.6 (range 0.2 - 31.4) years in the ELNSCS and 7.66 (range 0.12 - 19.96) years at UNIPD. Median age differed across treatment groups; HU group tended to be younger, while CTT group tended to be older. According to institutional standards of care, 47 (27.3%) patients from the ELNSCS and 50 (80.6%) from UNIPD received HU, 53 (30.8%) from the ELNSCS and 8 (12.9%) from UNIPD received CTT, and 72 (41.9%) from the ELNSCS and 4 (6.5%) from UNIPD received no treatment during the four year study period. Differences in treatment allocation reflect slightly different patient populations and approaches to care at the two centers. Severe VOEs persist among patients in all three treatment groups at both sites. Of those receiving HU, 83.0% from the ELNSCS and 68.0% at UNIPD had ≥1 severe VOE, including 21.3% from the ELNSCS and 44.0% at UNIPD experiencing ≥1 ACS event. The rate of severe VOEs in the HU group was 0.75 (range 0 - 39.5) per patient year from the ELNSCS and 0.49 (range 0 - 3.00) per patient year from UNIPD. HU dosing and adherence will be explored using data collected on hematologic parameters. In the ELNSCS, of those receiving CTT, 60.4% experienced ≥1 severe VOE (20.8% had ≥1 ACS event); of those receiving no therapy, 56.9% experienced ≥1 severe VOE (11.1% had ≥1 ACS event). At UNIPD, severe VOEs were observed in 62.5% of the CTT group and 50% of the no treatment group, though sample sizes were very small. C onclusions: Despite receiving expert care in accordance with local and international guidelines at two large academic centers, a significant sub-group of patients continue to experience severe VOEs. Results show that real-world usage of HU and CTT may not be optimized and, even if optimized, some patients may continue to experience severe acute complications including ACS. Both cohorts confirm that implementation of existing standard of care is insufficient to prevent significant morbidity in patients with SCD. Findings suggest the need to introduce DMT early in life to reduce and prevent acute complications and minimize disease progression. There is a persistent need for maximizing effective DMTs, as well as further developing curative therapies such as HSCT and gene therapy for both pediatric and adult patients. Figure 1 Figure 1. Disclosures Colombatti: Novartis: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BlueBirdBio: Membership on an entity's Board of Directors or advisory committees, Research Funding. Chawla: BlueBirdBio: Current Employment. Puri-Sharma: BlueBirdBio: Current Employment. Walls: BlueBirdBio: Current Employment. Kommera: BlueBirdBio: Current Employment. Telfer: Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Emmaus: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees; ApoPharma: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BlueBirdBio: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Terumo: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Neutrophil Extracellular Traps Are an Intrinsic Feature in Sickle Cell Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3651-3651
Author(s):  
Emilia Alina Barbu ◽  
Venina Marcela Dominical ◽  
Laurel Mendelsohn ◽  
James Nichols ◽  
Darlene Allen ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Fluorescence Microscopy ◽  
Sickle Cell ◽  
Acute Pain ◽  
Healthy Controls ◽  
Cell Disease ◽  
Extracellular Traps ◽  
Healthy Donors ◽  
Pain Crisis

Abstract Polymerisation of deoxygenated-HbS in sickle cell disease (SCD) leads to alteration of the shape and function of the red blood cells (RBC), resulting in hemolytic anemia and recurrent episodes of micro-vascular occlusion, triggering a cascade of downstream events and inflammation that underlies the progressive multi-systemic organ damage. Neutrophils have a critical role in promoting sickle pathophysiology; patients with higher leukocyte count, particularly neutrophils, tend to have a higher risk for more severe manifestations as well as earlier mortality. SCD neutrophils display a specific overreactive phenotype, including increased adhesive properties and production of reactive oxygen species. Activated neutrophils can also produce extracellular traps (NETs) in response to pathogens or other inflammatory stimuli. Studies have shown that prolonged exposure of healthy neutrophils to SCD plasma leads to formation of mature string-like NETs. These strings of decondensed chromatin covered with elastase (NE) or myeloperoxidase (MPO) might contribute to SCD pathophysiology. Here, we investigated if SCD neutrophils had enhanced intrinsic potential for NETs formation (i.e. could form NETs in the absence of a stimulus). We evaluated NETosis both indirectly, in which neutrophils isolated from healthy subjects were incubated with plasma from HbSS patients and directly, in which neutrophils isolated from HbSS patients were compared with those isolated from healthy donors. 17 HbSS patients in steady-state (all on hydroxyurea) and 11 race and sex matched healthy donors were included in the direct assays; the NETosis-related events were observed over a period of 7 minutes to 4 hours. For the indirect assay, we used plasma from 19 HbSS patients (14 on hydroxyurea, 5 off hydroxyurea and/or transfused), all of whom provided samples in steady-state and acute pain crisis (paired samples); the healthy neutrophils were treated with SCD plasma for up to 7 hours. A pain crisis was defined as an episode of acute pain that has no evident cause other than SCD, resulting in hospitalization and treatment with parenteral opioids. Steady state was defined as the period from at any time 8 weeks prior to or after a crisis. Patients were excluded when they were <18 or >80 years of age, pregnant or had a history of blood transfusion in the previous 8 weeks. The IRB approved the studies under NIH protocol numbers NCT00081523 (SCD subjects) and 03-H-0015 (healthy controls and SCD subjects). The NETosis features (histone citrullination H4cit3, nuclear decondensation, DNA-NE-MPO strings) were assessed by imaging flow cytometry and fluorescence microscopy. In the indirect assays, healthy neutrophils made more string NETs when treated with the SCD plasma (from both steady state and crisis) compared to plasma from healthy donors (Figure 1A), and NETs response was further increased with plasma from acute compared to their paired steady state sample (N=19, p=0.0114) (Figure 1B). Plasma from patients on or off hydroxyurea induced comparable NETosis responses. This confirmed a higher ability for the crisis milieu to modulate NETosis. Directly, neutrophils isolated from SCD patients as compared to those from healthy controls, showed increased number of decondensed H4cit3 positive nuclei, after only 7 min of incubation in RPMI (N=7, p=0.0688). By comparison the response to high concentration (20 uM) of Hemin - a regular product of hemolysis, was similar in the SCD and the healthy neutrophils (p=0.6871) (Figure 1C). This data suggested that the SCD environment might activate neutrophils to produce NETs in the absence of a specific stimulus. Fluorescence microscopy confirmed that neutrophils isolated from the patients did produce significantly more DNA-NE strings without any stimulation compared to healthy neutrophils (N=9, p=0.0079). After Hemin treatment, NETs response in SCD neutrophils was also higher than that of the healthy controls, but not statistically significant (Figure 1D). Our data provide the first direct in vitro evidence that neutrophils in SCD are innately prone to NETosis but whether and how these might contribute to the initiation or progress of SCD vaso-occlusive crises is yet to be determined. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differential Acetone Extraction of Total and Hemoprotein-Unbound Heme to Quantify Heme Binding Capacity of Plasma in Patients with Sickle Cell Disease: The Role of Heme Scavengers

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-15
Author(s):  
Madhav Vissa ◽  
Eric Soupene ◽  
Sandra K. Larkin ◽  
Lynne D. Neumayr ◽  
Elliott P. Vichinsky ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Research Funding ◽  
Binding Capacity ◽  
Principal Investigator ◽  
Heme Binding ◽  
Cell Disease ◽  
Intravascular Hemolysis ◽  
Free Hemoglobin ◽  
Neutral Ph

Introduction: Heme, an iron-containing protoporphyrin, is an essential component of hemoglobin that binds oxygen for delivery to tissues. In sickle cell disease, intravascular hemolysis leads to the presence of cell-free hemoglobin and heme, which may contribute to oxidative damage and activation of inflammatory pathways. Hemoproteins such as haptoglobin and hemopexin provide pathways to remove hemoglobin and heme, respectively, from circulation. Due to its hydrophobic nature, heme also intercalates in cell membranes and binds to plasma components such as albumin and lipoproteins, though with varying affinity. Hemopexin has high affinity for heme and removes heme from other heme pools in blood to counter the highly toxic properties of heme unbound to hemoproteins. Due to chronic hemolysis, hemopexin is depleted in individuals with sickle cell disease. We hypothesize that the reduction in heme binding capacity leads to increased unbound heme in blood and contributes to the pathogenesis of sickle cell disease. To define the different heme binding pools in patients with sickle cell disease, we developed a method requiring small amounts of plasma which allows measurement of total and hemoprotein-unbound heme. With this method, we can quantify the binding capacity of plasma for heme and correlate that with measurement of heme scavenging proteins. Methods: Blood from healthy individuals and sickle cell patients was collected in EDTA as anticoagulant under IRB approval. Plasma was separated by centrifugation from whole blood, and either processed fresh or after freezing at -80°C. Plasma protein was precipitated with a 4-fold volume of acetone at neutral pH (NA) or acidic pH (AA). Under acidic condition, heme is released from all heme binding pools, including hemoglobin, and provides detection of the total heme present in plasma. Under neutral pH condition, only heme unbound to plasma proteins is extracted. Once extracted, samples were dried and resuspended in DMSO. Heme concentration was spectrophotometrically determined at 400nm using standard curves prepared from hemin added in AA or NA. To determine heme binding capacity, hemin was added to serial dilutions of plasma and extracted in NA and AA as above. The appearance of heme in NA relative to AA represents the point at which heme binding capacity of plasma was saturated. This was compared to measurement of hemopexin and haptoglobin using commercially available ELISA measurements. Hemopexin and albumin were added to samples to modulate heme binding capacity. Results: Heme concentration closely correlates with spectroscopic measurement of heme in DMSO confirming reliable quantification of total and unbound heme in acidic and neutral acetone extractions as low as 2.5µM. We next show that heme binding capacity can be determined. Heme added to plasma was effectively recovered in AA extracts and begins to appear in the NA extract when binding sites start to become saturated. We note that not all sites appear to be fully saturated before heme is detected in NA extract. Addition of hemopexin to plasma increased the binding capacity on an equimolar basis, indicating that hemopexin effectively binds heme present in plasma. In samples from patients with sickle cell disease, concentration of total and unbound heme varied widely, and did not necessarily correlate with degree of intravascular hemolysis, estimated based on the measurement of cell free hemoglobin. Both the capacity of plasma to bind heme and levels of hemopexin indicated that, in a number of patients, the amount of heme present was greater than the ability of hemopexin to bind cell free heme. Discussion: We present a novel method to quantitatively differentiate hemoprotein-bound and unbound heme in plasma, the latter of which is pathologically relevant in sickle cell disease. Our data show significant variation in the concentration of total and unbound heme in sickle cell patient samples, and that the binding capacity in sickle cell plasma only partially correlates to the degree of hemolysis measured based on cell free hemoglobin. Patients are currently enrolled in a clinical study to measure intra-patient differences in heme and heme-binding capacity during steady state and during acute sickle cell-related illness. Understanding the clinical implications of heme and heme scavengers may provide insights into diagnostic and therapeutic targets for patients with sickle cell disease. Disclosures Neumayr: Emmaus: Consultancy; Bayer: Consultancy; CTD Holdings: Consultancy; Pfizer: Consultancy; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Micelle: Other: Site principal investigator; GBT: Other: Site principal investigator; PCORI: Other: site principal investigator; Novartis: Other: co-investigator; Bluebird Bio: Other: co-investigator; Sangamo Therapeutics: Other; Silarus: Other; Celgene: Other; La Jolla Pharmaceuticals: Other; Forma: Other; Centers for Disease Control and Prevention: Other; Seattle Children's Research: Other; Imara: Other; National Heart, Lung, and Blood Institute: Other; Health Resources and Services Administration: Other. Vichinsky:Bluebird Bio: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding; GBT: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Kuypers:Forma Therapeutics, Inc.: Research Funding.

Elevated Circulating Stromal Derived Factor-1 Levels in Sickle Cell Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1438-1438
Author(s):  
Precious Landburg ◽  
Erfan Nur ◽  
Naomi Maria ◽  
Bart J. Biemond ◽  
Dees P.M. Brandjes ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Organ Damage ◽  
Cell Disease ◽  
Steady State Levels ◽  
Leukocyte Counts ◽  
Painful Crisis

Abstract Inflammation and angiogenesis are of importance in the pathophysiology of sickle cell disease (SCD). Recently, the chemokine Stromal Derived Factor-1 (SDF-1) has been shown to be a key mediator of angiogenesis and inflammation. In this study we determined serum SDF-1 levels in consecutive adult sickle cell patients during the clinically asymptomatic state as well as during painful crisis. Serum SDF-1 levels were significantly elevated in HbSS/HbSβ0-thalassemia patients ([n=39], 3805 pg/mL (2121–6790)) as opposed to HbSC/HbSβ+-thalassemia patients ([n=18], 2405 pg/mL (1365–3047)) and healthy HbAA controls (n=25, 2623 pg/mL (2435–2988)) (p=0.04). During painful crisis SDF-1 levels increased significantly in both HbSS/HbSβ0-thalassemia ([n=24]) 26040 pg/mL (20135–27960)) and HbSC/HbSβ+-thalassemia patients ([n=5], 12666 pg/mL (3936–24453)) (p&lt;0.001 and p=0.01, respectively). Paired sample analysis was possible in 8 patients (SDF-1 determined in steady state and during painful crisis) and all patients demonstrated a strong SDF-1 increment from steady state levels (p=0.01) and serial measurements, available from 11 patients, showed persistently elevated SDF-1 levels well beyond crisis abatement (data not shown). Furthermore, SDF-1 levels were significantly higher in sickle cell patients with pulmonary hypertension as compared to patients without pulmonary hypertension in both HbSS/HbSβ0-thalassemia (PHT+ [n=8]: 6549 pg/mL (3893–7159), PHT- [n=24]: 2945 pg/mL (1560–6627)) and HbSC/HbSβ+-thalassemia (PHT+ [n=4] 3316 pg/mL (2900–6417) and PHT- [n=12] 2146 pg/mL (1138–2592)) patients (p=0.02 and p=0.01, respectively). SDF-1 levels were not related to other forms of organ damage, nor were they significantly associated with hemoglobin levels, the percentage of fetal hemoglobin or leukocyte counts in the clinically asymptomatic state (data not shown). Taken together, elevated circulating SDF-1 levels occur in patients with SCD and may play a role in the pathophysiology of acute and specific chronic disease related complications.

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