Vaso-occlusive Episodes Research Articles

Dietary Iron Restriction Alters Microbiota-Host Crosstalk and Protects Against Vaso-Occlusion and Organ Damage in Sickle Cell Disease Mice

Introduction: Sickle cell disease (SCD) is the most commonly inherited monogenic hemoglobinopathy and results from a mutation in the β-globin gene (HbS), leading to red blood cell (RBC) sickling, vaso-occlusive episodes (VOE), and organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron-overload, and another subpopulation of SCD patients manifest iron deficiency. SCD mice exhibit increased iron absorption via upregulation of the intestinal transcription factor hypoxia inducible factor-2a(HIF-2a) leading to the development of iron overload. Disruption of intestinal HIF-2a or limiting iron intake by dietary iron restriction in SCD mice, significantly reduced systemic iron overload, lowered cellular sickle hemoglobin, and consequently improved hemolysis and ameliorated anemia. Our lab previously demonstrated that microbial signals from the gut also participated in initiation of VOE and organ damage in SCD mice. To elucidate connections between dietary iron, the microbiota-gut axis, and SCD pathogenesis, we hypothesize that dietary iron restriction may reduce microbiota load and gut permeability, and contributes to amelioration of acute and chronic complications in SCD mice. Methods: SCD mice were generated by transplanting bone marrow cells from Berkeley SCD (Tg[Hu-miniLCRα1GγAγδβS]Hba−/− Hbb−/−) mice into 8-week old, lethally-irradiated C57BL/6J mice. Immediately after transplantation, SCD mice were fed either a regular-iron diet (Ctrl) containing 185 parts per million (ppm) iron or an otherwise identical iron-restricted diet (IRD) containing 3 ppm iron. VOE and organ damage analysis were conducted 3-4 months after treatments. Results: IRD resulted in a reduced organ iron concentration in SCD compared to Ctrl group(0.6±0.1 v.s. 4.0±0.7ug iron/mg liver tissue; P=0.001), confirming that dietary iron restriction reverses organ iron deposition in SCD mice. SCD mice treated with IRD exhibited reduced MCHC (19.7±0.3 v.s. 20.1±0.2 pg; P=0.006) and serum indirect bilirubin (0.4±0.04 v.s. 0.7±0.07 mg/dL; P=0.001) indicating a decrease in cellular sickle hemoglobin and hemolysis. Furthermore, IRD reduced the fraction of aged neutrophils by >50% in SCD mice which corresponded with improved VOE parameters (RBC velocity: 2.0±0.2 v.s. 1.6±0.1mm/s; P=0.01; blood flow rate: 314±33 v.s. 230±13pL/s; P=0.01; leukocyte adhesion: 10±1 v.s. 14±1cells/100um vein; P=0.01; IRD v.s. Ctrl). Importantly, IRD led to prolonged survival after TNF-a challenge in SCD mice (Fig1). IRD resulted in a dramatic reduction in lung leukocyte infiltration, indicating significantly reduced lung inflammation in SCD mice (Fig2A). Compared to Ctrl liver samples, IRD resulted in significantly less liver necrosis, leukocyte infiltration and fibrosis (Fig2B). We further hypothesized that, in addition to reducing iron concentration in organs, IRD ameliorates organ damage by preventing translocation of microbial compounds. Microbiota load in IRD treated SCD mice were decreased to 25% of Ctrl level. IRD inhibited translocation of FITC-Dextran by 5-fold compared to Ctrl suggesting functionally improved gut permeability. We detected significantly decreased TLR2 ligands in serum of IRD compared to Ctrl mice (0.31±0.04 v.s. 0.46±0.05 TLR2 ligand activities O.D. 650nm; P=0.04) suggesting ligands from gram-positive bacteria (TLR2 agonists) may play a role in triggering SCD complications. Conclusion: Our study demonstrates first time that dietary iron restriction reduces VOE severity and alleviates chronic organ damage in SCD mice. These findings provide strong evidence for the therapeutic potential of manipulating iron homeostasis and the gut microbiome to ameliorate SCD pathophysiology. Many treatments under development focus on lowering the systemic iron concentration to relieve disease complications, and our data suggest that iron-induced changes in microbiota load and gut integrity are related and novel therapeutic targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

"It's Kind of a Hidden Code": An Exploratory Study of Residents’ Experiences Caring for People with Sickle Cell Disease on the Internal Medicine Wards

introduction: Sickle cell disease (SCD) hospitalizations are rising in Canada. Painful vaso-occlusive episodes (VOEs) are responsible for 90% of acute health care visits for patients with SCD and are often recurrent with more than 50% of patients being re-admitted in the first month after discharge. Yet, there is little medical education literature on SCD and pain management. Also, while it is known that anti-black racism leads to poorer healthcare outcomes for SCD patients, there is no literature on how this impacts medical education, particularly when it comes to internal medicine residents. Objectives: To determine the training experiences and gaps in knowledge, skills, and attitudes of internal medicine residents who care for patients with SCD suffering from VOEs. Methods: We conducted a qualitative study using in-depth, one-on-one interviews with internal medicine residents at the University of Toronto. A semi-structured interview guide was iteratively developed by the study team to explore topics around experiences caring for patients with SCD, the perception of bias in in-patient care for SCD, and training and education around SCD and VOEs. Participant demographics were also collected (post-graduate year, gender, race, and number of patients with SCD cared for). Both purposive and snowball sampling were used for recruitment. All interviews were recorded and professionally transcribed. Qualitative content analysis guided the analysis phase. Codes were developed inductively and discussed between three coders (FK, WY, JZ) until consensus was reached. Interview memo notes were also incorporated in the analysis to capture body language and context that may be missed in the transcription. After four interviews, coders began reading transcribed interviews to conduct analysis. Concurrent analysis allowed the research team to look for thematic saturation. Research ethics approval was obtained. Results: Twelve interviews were completed. Five participants identified as female, and seven as male. Most participants (5/12) were in their post-graduate year 2, and 10/12 participants described having significant exposure to patients with SCD (>3 patients per year). Participants self-reported race included 3/12 South Asian, 3/12 Arab, 2/12 Caucasian, 1 Asian, 1 Black, amongst others. Preliminary findings after analyzing 5 transcriptions are summarized in Table 1. Patients with SCD are treated differently than other in-patients for their care, whether this is operationalized through an unspoken hidden code within healthcare providers or through racial biases. For many internal medicine residents, trust between them and their patient with SCD is paramount: patients with SCD have historically distrusted physicians and the healthcare system. Some patients, however, are still willing to listen to their counselling physicians and allow residents to change their care plan. There is little SCD and pain education taught during residency and much of pain management is learned from cancer or palliative pain. Residents remain feeling uncomfortable managing the pain of patients with VOEs. Conclusion: While these are preliminary findings, they support a wider literature that demonstrates inadequate SCD education during medical training. This study was completed in the largest SCD centre in Canada and yet, educational deficiencies were still noted amongst rotating internal medicine residents suggesting that they are widespread. We hope to use these findings to develop curricular recommendations for training in the management of VOEs, implicit bias training of internal medicine residents, as well as develop teaching moments around health care disparities and societal inequities regarding delivery of care to individuals with SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Assessment of Emergency Department Care for Patients with Acute Pain Crisis after the Formation of a Multidisciplinary Team

Introduction:American Society of Hematology (ASH) guidelines recommend the rapid evaluation and treatment of patients presenting to the Emergency Department (ED) with pain from an acute vaso-occlusive episode (VOE) related to sickle cell disease (SCD). The treatment of severe pain from VOE in SCD is time dependent and time sensitive. Guidelines recommend that treatment for acute VOE with parental opioid analgesia occurs within 60 minutes of ED registration. With respect to the five-level Emergency Severity Index (ESI) triage tool, in which lower numbers indicate higher acuity, patients with SCD are to be assigned an Emergency Severity Index (ESI) of 2 indicating (a) greater disease burden, and (b) the need for high priority bed assignment. Objectives: We aimed to create a multidisciplinary team of providers to assess and address the existing barriers and facilitators to improving guideline adherence for the management of acute crises in our large academic health system. The primary outcome of this initiative was to improve guideline adherence by ensuring 90% or greater adherence to ESI 2 assignment to acknowledge illness severity as well as timely, within 60-minute opioid analgesia. Methods: In March 2020, we convened a multidisciplinary team of stakeholders to discuss the ED care of patients with SCD presenting with an acute VOE. The group comprises ED and infusion center nurse champions, nurse educators, nurse leaders, hematologists, palliative care specialists, primary care physicians, psychologists, advanced practice providers and other allied health professionals including social work and spiritual care. Initial monthly meetings established recommendations based on national guidelines and discussed interventions to improve guideline adherence including 1. a nursing driven pain management orderset with subcutaneous and intranasal opioids, 2. a best practice alert with the recommended guidelines. We conducted a retrospective review of ED visit data including all patients with a known diagnosis of sickle cell disease presenting to the ED with a chief complaint of acute pain related to VOE from January 2018-February 2020 and March 2020 to March 2022. We compared the before and after intervention using descriptive statistics. Results: Of the 760 unique encounters for acute pain related to sickle cell disease from 2018-2022, 406 visits occurred before the group formation and 354 visits occurred after the group formed. Mean age was 30.5 years (+/- 19.5) in the pre-intervention period compared to the 29.5 years (+/- 18.9) in the post intervention period. The time to the first opioid order placed pre-intervention averaged 53.3 mins (+/- 64.9) was not significantly different compared to the post period 75.2 mins (+/- 125.8; p =0.17). The proportion of patients assigned an ESI 2 changed from 49.3% to 50.5%. Conclusion: While there was a marginal increase in the ESI 2 assignment among SCD patients presenting in acute VOE, there was no significant change in the ED time to first opioid after the formation of a multidisciplinary team.

Emergency Department Experience and the Use of Non-Pharmacologic Treatment for Acute Pain Among Patients with Sickle Disease

Background: Appropriate management for patients presenting to the Emergency Department (ED) with acute pain from vaso-occlusive episodes (VOE) primarily includes treatment with parenteral opioid and non-opioid analgesia and fluids if the episode is uncomplicated. Non-pharmacological strategies that significantly affect non-physical aspects of behavioral, social, and cultural life, have been studied for pain treatment in sickle cell patients; however, the data on the effectiveness of these methods are limited. Objectives: This work aims to assess the overall ED experience among patients seeking care for acute VOE. We also aim to assess any effective alternative pain management strategies used by patients to treat their acute pain symptoms. Methods: We conducted ED follow up assessments for patients who sought care in the ED for acute pain from vaso-occlusive episodes from January 1, 2022- July 15, 2022. These were brief individual phone interviews. The semi-structured interview guide was created using a hospital specific patient experience questionnaire in addition to questions pertaining to pain management in the acute and day-to-day settings, demographic data including, age, sex, emergency department visit characteristics (length of stay (LOS), emergency severity index assignment (ESI), time to first opioid). The interviews were analyzed using a framework matrix analysis. This qualitative data reduction technique was used to review, summarize, and classify the interview data. NVivo 20 qualitative data analysis software includes a framework matrix tool used to manage data and facilitate this analysis in which participant comments about key content were aggregated and entered for review. Results: Overview: Among the 21 patients who met study criteria, eight participants are included in this analysis. Six participants identified as male, and two participants identified as female. ED Experience: Patients rated their ED care from a 3/10 to a 10/10 (avg 6.4, +/- 2.6). Common themes included prolonged wait times for pain management (6/8 participants). Five of the eight patients interviewed expressed gratitude for the care provided though noted some room for improvement. One participant noted "they could have been nicer to me. It's like they don't talk to each other" (2/8 participants). Patients also expressed frustration when intravenous access issues arise and compounding delays in treatment. Pain management: Three of the eight patients interviewed reported using scheduled opioids (oxycodone-5 patients, and methadone-2 patients) at home every 3-8 hours. One patient declined to answer questions about home opioid use. All patients received hydromorphone for acute management in the ED. Two of the eight patients expressed knowledge of their care coordination pain management plan. Alternative pain management: Five of the eight patients used heat or hot packs as their primary alternative pain management and specifically referenced this as an intervention that they would find helpful in the ED. Two patients prioritized distractions such as video games or other screen time. One patient relied on family support, massages, and meditation. Conclusions: Patients experiencing acute pain from VOE rely on the ED for care. Optimizing that care will not only improve patient's health, but also their well-being. Considering complementary alternatives for acute pain management including heat therapy (packs, blankets) as well as massage (heated chair) or distractions (video games, reading or movies) may provide additional benefits while patients seek care for acute pain crises.

Subcutaneous Injection of IHP-102 Attenuates Lung Vaso-Occlusion in Sickle Cell Disease Mice

Sickle Cell Disease (SCD) is an autosomal-recessive-genetic disorder that affects ~100,000 Americans and over 3 million people world-wide. Acute systemic painful vaso-occlusive episode (VOE) is the primary reason for hospitalization of SCD patients and serves as an antecedent to acute chest syndrome (ACS), a type of acute lung injury and one of the primary reasons for mortality among SCD patients. Intravenous (IV) injection of P-selectin monoclonal antibodies have been shown to attenuate lung vaso-occlusion by neutrophil-platelet-erythrocyte aggregates in transgenic-humanized SCD mice and reduce frequency of VOE in SCD patients. However, IV route of administration requires at least an out-patient visit to the clinic, thus highlighting the need for therapies that can cut-down the health care cost by enabling self-administration (such as sub-cutaneous) by SCD patients at home during the prodromal phase of a VOE. Importantly, P-selectin dependent neutrophil-platelet aggregation and the complement pathway activation contribute to the vaso-occlusive pathophysiology in SCD. IHP-102 is a novel compound, which serves as a dual-inhibitor of both P-selectin and the complement pathway. Our initial pharmacodynamic findings in rodents revealed that P-selectin-inhibition activity of IHP-102 was detectable in the serum within 30 min following sub-cutaneous administration of 30 mg/kg IHP-102. Next, quantitative fluorescence intravital lung microscopy (qFILM) was conducted to assess the efficacy of IHP-102 in attenuating intravenous 10 μmole/kg oxy-hemoglobin (IV oxy-Hb) triggered lung vaso-occlusion in Townes SCD mice. Remarkably, and despite the described vasculopathy that would impair systemic delivery in this model, sub-cutaneous administration of either 10 or 30 mg/kg IHP-102 led to significant attenuation of lung vaso-occlusion by neutrophil-platelet aggregates in Townes SCD mice administered IV oxy-Hb. These findings are the first to highlight the therapeutic potential of a subcutaneously administrable therapy (IHP-102) in preventing VOE and ACS in SCD.

Impact of Arginine Therapy on Clinical Outcomes, Mitochondrial Function and Oxidative Stress in Children with Sickle Cell Disease Hospitalized with Vasoocclusive Pain Episodes: A Randomized Controlled Trial

Introduction: Vaso-occlusive pain episodes (VOE) are the leading cause of emergency department (ED) visits & hospitalization in patients with sickle cell disease (SCD). During SCD-VOE, patients develop an acute arginine (Arg) deficiency. Supplementation with Arg has shown opioid-sparing effects, improves blood pressure & cardiopulmonary function & decreases length of hospital stay. Though we have previously demonstrated that intravenous (IV) Arg improves mitochondrial (mito) function, the precise mechanisms by which Arg mediates these positive effects remain unclear. Objectives: To determine the role of IV Arg as adjuvant in the management of SCD-VOE Methods: Prospective single-center double-blind randomized controlled trial (RCT) of IV Arg (TID, up to 7 days) in children with SCD age 3-21 years hospitalized for VOE requiring IV opioids. Patients with significant liver/renal dysfunction or those previously enrolled were excluded. Subjects were randomized into 1 of 3 arms: 1) 100 mg/kg/dose Arg, standard dose (SD), 2) loading dose: 200 mg/kg followed by SD or 3) placebo. Demographics, total parenteral opioid (TPO) use (morphine equivalents, mg/kg) time to crisis resolution (time of study drug delivery to last IV opioid in hours), pain scores, patient reported outcomes (PROMIS) & mito function were obtained before treatment & at discharge. The primary outcome measure was TPO use between study arms. Hypothesis tests & unadjusted & covariate-adjusted fixed & mixed effects generalized linear regression models were used, as appropriate, to compare cross-sectional & longitudinal outcomes within & between randomization arms. This protocol utilized IND#66943 (Sponsor-Morris), registered with ClinicalTrials.gov (NCT02536170). Results: 1,548 patients were screened, 266 were eligible, 114 consented, & 108 were randomized. Safety results of this RCT have been previously reported (Reyes et al, Am J Hematol 2022). Demographics & clinical outcomes of subjects randomized by treatment arm are provided in Table 1. While statistically insignificant, there was a clinically relevant decrease in TPO & time to crisis resolution in both Arg arms compared to placebo. The placebo group required 45% higher TPO & experienced >15 hours longer mean time to crisis resolution compared to combined Arg-treated groups when adjusted for HU use, continuous age and sex. Among children <17 years, the placebo group (n=33) required 80% more TPO compared to combined Arg groups (n=57; p=0.075). No differences in ED vs discharge pain scores or patient/parent PROMIS reports across arms were found. Mito Complex V activity was higher (p=0.02) & protein carbonyl levels were lower (p=0.003) at ED visit in patients on Hydroxyurea (HU). Notably mito Complex IV & V activity increased significantly in both Arg arms while there was no change in the placebo group (Fig 1, p<0.001); protein carbonyl levels in platelet rich plasma decreased in both Arg groups (Fig 1, p<0.001), suggesting a decrease in oxidative stress that increased in the placebo arm (p=0.02). Greatest mito improvement occurred with Arg loading dose. Conclusion: Arg supplementation increases mito activity & decreases oxidative stress in children with SCD-VOE. This is the first report to suggest a HU-related impact on mito activity in SCD. Prior reports show that Complex V inhibition leads to increased mito oxidant production in platelets from SCD patients. Acute improvements in Complex IV & V function & decreased oxidative markers with Arg therapy are consistent with Arg-induced reductions in mito oxidant generation. Arg therapy improves symptoms associated with MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) suggesting an impact of Arg on mito activity that may be beneficial in SCD. Given emerging data supporting a link between mito function and pain, improved mito activity may mechanistically contribute to decreased pain & ultimately less opioid requirement with IV Arg treatment, as well as have further implications for improved metabolism and oxidative signaling in SCD. In this RCT, Arg therapy also demonstrated clinically relevant opioid-sparing & a decreased time to crisis resolution that was statistically insignificant. A larger sample size may elucidate potential differences; we are currently enrolling 360 children in an ongoing PECARN-endorsed multicenter Phase 3 RCT (SCD Treatment with Arg Therapy - STArT). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lnp-Targeting Hematopoietic Stem Cells and Lentiviral Gene Transfer to Generate and Rescue a Novel Mouse Model of Lethal Alpha-Thalassemia

α-Thalassemia (α-thal) is caused by insufficient production of the α-globin protein. Clinical presentation of α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to Bart's Hydrops Fetalis Syndrome/BHFS (complete knockout). In patients with severe α-thal, allogeneic bone marrow transplantation may be required for survival. Alternatively, patients could be treated through autologous BMT following ex vivo transduction with a human α-globin expressing lentiviral vector. However, a proper model system and vector must be established for the development of such a treatment. We crossed animals that had deletion of both Hba-a1 and Hba-a2 in heterozygosity (Hba-a1+/ko/Hba-a2+/ko) to generate knockout (KO) embryonic mice lacking all the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko). Although these embryos die perinatally, their fetal liver cells (FLC) at 13.5 dpc are still viable and represent a source of engraftable hematopoietic stem cells (HSC). KO-FLC were transplanted into congenic myeloablated WT mice to generate mice carrying Hba-a1ko/ko/Hba-a2ko/ko HSC. These animals developed a severe phenotype ~7 weeks post engraftment. As these mice lack α-globin expression, we observed β-tetramer formation (β4, HbH, Fig1A), resulting in aberrant red blood cell (RBC) morphology, β-globin precipitates, high RBC oxygen binding affinity, elevated erythropoietin, hematocrit and hemoglobin levels, splenomegaly and vaso-occlusive episodes, due to the large number of abnormal RBCs. Furthermore, we observed iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver. However, FLC engraftment is cumbersome and requires extensive breeding and many animals to generate enough FLC for few recipients. To facilitate the generation of α-thal mice, we created conditional knockout (cKO) mice that have been edited to remove Hba-a2, while inserting loxP sites flanking Hba-a1 (Hba-a1fl/Hba-a2ko). Homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) demonstrate a mild phenotype. To achieve complete deletion of the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko) in HSC, we used lipid nanoparticles (LNPs), which are one of the most promising delivery systems to package and make mRNA into a useful therapeutic. We developed a novel LNP preparation with an antibody to target CD117 and deliver nucleoside-modified mRNA to HSC, (see abstract Breda et al). We treated HSC from homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) ex vivo with CD117-LNP embedded with Cre mRNAs (Cre-CD117-LNP). Cells showed the expected deletion of the α-globin genes and were injected into myeloablated recipient mice. These chimeras recapitulated the phenotype observed following transplantation of constitutive α-globin-KO FLC, including elevated hematocrit, splenomegaly, and culminating in lethality ~7 weeks post-transplant. We screened multiple potential erythroid specific lentiviral vectors in both mouse and human cell lines and identified a promising candidate, ALS20αI, which at VCN=1 produced human α-globin protein equivalent to that of a single endogenous α-globin gene. Myeloablated recipient mice transplanted with Cre-CD117-LNP-treated cKO BM die roughly 7 weeks post-transplant with the expected pathological phenotype (Fig1A). In contrast, mice receiving BM treated with Cre-CD117-LNP and ALS20αI (VCN>1) all survived long term (>4-month) with normalization of erythropoiesis and iron metabolism. VCNs were proportional to chimeric hemoglobin (human α/mouse β) levels and concurrently decreased or absent HbH (Fig1B-C-D). Additionally, we tested ALS20αI in erythroid progenitors derived from CD34 cells isolated from patients with deletional and non-deletional HbH disease. Preliminary data demonstrates improvement in α-globin/β-globin mRNA ratio and reduction in formation of HbH by HPLC. In summary, we have developed new mouse models for α-thal with a lethal and reproducible phenotype. Remarkably, use of LNP technology accelerated the generation of α-thal mice and their characterization. Furthermore, our new vector ALS20αI rescued these animals with stable expression of the human α-globin gene. These data strongly support the use of ALS20αI for the cure of severe forms of α-thal, as 2 copies may be sufficient to rescue patients affected by BHFS, while 1 copy may be sufficient to cure patients affected by HbH disease. HP, SR corresponding Authors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lovo-cel (bb1111) Gene Therapy for Sickle Cell Disease: Updated Clinical Results and Investigations into Two Cases of Anemia from Group C of the Phase 1/2 HGB-206 Study

Background: Lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) uses autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) transduced with the BB305 lentiviral vector encoding a modified β-globin gene, which produces an anti-sickling hemoglobin (Hb), HbAT87Q. The ongoing phase 1/2 HGB-206 study (NCT02140554) is the largest clinical trial of GT in SCD to date. We report updated efficacy and safety results for Group C and describe the results of additional investigations in two patients (pts) in Group C with persistent anemia. Methods: Pts (≥12-≤50 yrs of age) with SCD and recurrent severe vaso-occlusive episodes (VOEs) underwent plerixafor mobilization and apheresis followed by myeloablative busulfan conditioning and lovo-cel infusion. Lab evaluations, SCD-related outcomes, and safety were assessed. Data are median (min-max) unless otherwise stated; all timepoints are from lovo-cel infusion. Results: As of July 2021, 35 Group C pts had received a lovo-cel infusion, with a median follow-up of 20.9 months (min-max: 8.5-28.5). Neutrophil and platelet engraftment were achieved on Days 20 (12-35) and 36 (18-136), respectively. The median vector copy number in peripheral blood remained stable in all pts at ≥1 c/dg from 6 months after infusion until the last study visit, and resulted in median HbAT87Q levels of ≥5 g/dL during this period. The median total Hb level increased without transfusions from 8.5 g/dL at baseline to ≥11 g/dL from 6 months through last visit; HbAT87Q contributed at least 40% of total Hb (Fig 1A). Of the 29 pts who could be evaluated, 28 had a complete resolution of severe VOEs after lovo-cel infusion in the 2-yr parent study as of last follow-up, as compared with 3.5 events per yr (1.0-13.5) in the 2 yrs before enrollment (Fig. 1B); no severe VOEs have been reported during the long-term follow up study. Key hemolysis markers approached normal levels. Overall, 15 pts (43%) had ≥1 serious adverse event after lovo-cel infusion; the most frequently reported were abdominal pain, drug withdrawal syndrome (opiate), nausea, vomiting, and dehydration (6% each). No cases of veno-occlusive liver disease, graft failure, replication-competent lentivirus, vector-mediated insertional oncogenesis, or hematologic malignancies were observed. The lovo-cel treatment regimen generally reflected the known side effects of HSPC collection and busulfan conditioning regimen, and/or adverse events commonly seen in the population being evaluated. Further investigations were conducted as of March 2022 in two pts who presented with similar clinical and pathological findings that initially raised suspicion of myelodysplastic syndrome (MDS). Presentation features included neutropenia in one patient; both patients had persistent anemia despite adequate HbAT87Q production, bone marrow morphology showing erythroid specific dysplasia (binucleated or trinucleated cells) consistent with dyserythropoiesis as seen in other hemoglobinopathies, and low level of trisomy 8 (≤7.7%) only observed by fluorescence in situ hybridization analysis. There are no clinical symptoms suggestive of hematologic malignancies, no driver mutations, no clonal process (vector-related or otherwise) as shown by Integration Site Analysis and Next-Generation Sequencing (both subjects had >29,000 Unique Integration Sites with no integration having a relative frequency above 0.425%), and no chromosomal abnormalities on karyotyping among up to 205 metaphases. Both pts have had anemia and one maintained total Hb of 9.8 g/dL at Month 18 without transfusions, and the other required chronic transfusions to maintain 10.3 g/dL at Month 18 with otherwise normal complete blood counts. Both pts experienced a reduction or resolution of severe VOEs after lovo-cel infusion. Of note, these are the only pts in the study to have two α-globin deletions (−α3.7/−α3.7). We propose that α-globin trait may contribute to anemia and dyserythropoiesis after lovo-cel infusion. Summary: In Group C of the HGB-206 study, one-time treatment with lovo-cel resulted in sustained production of HbAT87Qand near-complete resolution of severe VOEs. Investigations to date in both patients with anemia have not confirmed MDS; thus, currently no hematologic malignancies have occurred in Group C. Ongoing long-term follow-up should be continued for all pts receiving GT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prediction of High Acute Care Utilization in Children with Sickle Cell Disease

Background: A small percentage of patients with sickle cell disease (SCD) account for a high percentage of acute care utilization and painful vaso-occlusive episodes (VOEs) requiring hospitalization. Chronic pain may develop in patients with SCD who experience frequent VOEs. Little is known about the mechanisms mediating the development of chronic pain in SCD, and there are no reliable clinical predictors for the development of a chronic pain phenotype. We aimed to determine the factors associated with the development of chronic pain phenotype characterized by high acute care utilization, in children and adolescents with SCD. Methods: We performed a single-institution, retrospective case-control study of all patients aged 10-25 years old who received SCD care at our institution from 2016-2020. Patients were identified using ICD-10 codes for SCD. For patients meeting inclusion/exclusion criteria, we extracted demographic, laboratory, and opioid prescription data. ED visits and hospitalization encounters during which parenteral opioids were administered were identified in the electronic medical record. Distribution of acute care encounters among all included patients was used to establish cases who met criteria for high acute care utilization, defined as > 85th percentile for all patients. Laboratory data were collected from the index year, defined as the first year a case met criteria for high acute care utilization, or from the matched year within 1-2 year window, for controls, as well as from the year prior. Chi-square and student t test were used to compare categorical and continuous variables, respectively, between cases and controls using SAS (Cary, NC). ROC curves were created to determine the discriminatory ability of using opioid prescription data to identify patients meeting criteria for high acute care utilization. Results: A total of 200 patients with SCD (all genotypes) met inclusion/exclusion criteria for the study. The 85thpercentile cut point corresponded to ≥4 acute care encounters in one year. Using this definition, 36/200 (18%) patients who were considered "high acute care utilizers" and had ≥4 acute care visits during any 1-year period during 2016-2020 were chosen as cases. Matching 2:1 by genotype and calendar year, 72 "low-utilizers" were randomly selected as controls among the 127 patients who had ≤1 acute care visit during each year of inclusion. Age, sex, and hydroxyurea use did not differ among cases vs controls. We found a higher proportion of patients with Medicaid insurance (83% vs 62%, p=0.03), as well as asthma (33% vs 11%, p=0.008) and anxiety or depression (39% vs 2%, p<0.001), among cases vs controls. Steady-state laboratory variables did not differ significantly among cases vs controls. Total opioid prescriptions were significantly higher in both index year [7.7 (95% CI: 5.2-10.2) vs 1.3 (0.9-1.7), p<0.001] and in the year prior [4.6 (95% CI: 1.9-7.2) vs 1.0 (0.7-1.3), p=0.01] among cases vs controls. Total morphine equivalents during the index year utilizers [3540 (95% CI: 1010-6060) vs 554 (355-753), p=0.02] and in the year prior [1386 mg (95% CI: 597-2170) vs 448 mg (249-648), p=0.01] were significantly higher among high acute care utilizers. ROC curves for average total opioid prescriptions and total morphine equivalents prescribed in the year prior to meeting criteria for high acute care utilization demonstrated AUCs of 0.80 and 0.81, respectively. Using these ROC curves, a cutpoint of ≥2 opioid prescriptions in the prior year was associated with a maximal sensitivity of 75% and a specificity of 76%. A cutpoint of ≥400mg of total morphine equivalents in the year prior was associated with a maximal sensitivity of 81% and a maximal specificity of 71%. Conclusions: We found no relationship between laboratory variables and high acute care utilization in children and young adults with SCD. Demographic and clinical factors associated with high acute care utilization include Medicaid insurance status, asthma and mood disorders as well as number of opioid prescriptions in the year prior to meeting our criterion for high acute care utilization. Future studies should be conducted to prospectively identify predictors of high acute care utilization and the development of chronic pain in SCD. The role of opioids for the treatment of chronic pain in SCD and the relationship of opioid to the development of high acute care utilization and chronic pain should also be further studied.

The Role of TSP-1, TGF-B1 and Galectin-3 in the Pathogenesis of ACS in Sickle Cell Disease

Sickle cell disease (SCD) is a hereditary blood disorder characterized by painful vaso-occlusive episodes (VOE), acute chest syndrome (ACS), stroke and other clinical complications. Seventy-six percent of the primary and secondary complications of SCD are pulmonary and are strongly associated with hemolysis. ACS is the main acute pulmonary complication of SCD and is characterized by critical lung injury from vaso-occlusion in the pulmonary vasculature, release of inflammatory mediators, and alveolar edema decreasing gas exchange. Endothelial dysfunction, characterized by increased production of inflammatory mediators, increased oxidative stress, and aberrant cellular proliferation via platelet-derived thrombospondin-1 (TSP-1) and transforming growth factor-1 (TGF-B1) is increasingly implicated in pulmonary vascular pathology in other diseases. In addition, a mediator of TGF-B1, galectin-3, has been shown to be induce tissue-specific fibrosis via fibroblast activation in animal disease models. However, the mechanisms by which human pulmonary artery endothelial cell (HPAEC) dysfunction is instigated remains unclear. We hypothesized a novel theory that TSP-1, TGF-B1, and galectin-3 play a causal role in the pathogenesis of ACS in SCD. We set out to test our hypothesis by measuring TSP-1, TGF-B1 and galectin-3 plasma levels in a cohort of patients with SCD with (n=10) and without history of ACS (n=20), and healthy, race and age-matched controls (n=10). Patients with ACS had significantly higher plasma levels of active TGF-B1 (11.34 ng/mL) than those of patients without ACS (5.2 ng/mL) and healthy controls (3.4 ng/mL). Similarly, we found that SCD patients with a history of ACS had significantly higher levels of TSP-1 (642.7 ng/mL) and galectin-3 (8.87 ng/mL) as compared to patients without ACS (285.9 ng/mL and 6.02 ng/mL, respectively) and controls (133.36 ng/mL and 5.2 ng/mL, respectively). We also found a significant correlation between TSP-1 and TGF-B1 levels in non-ACS SCD patients and patients with ACS as well as galectin-3 and TGF-B1 levels in the plasma of all SCD patients combined. Given that platelets are a significant source of TSP-1 and TGF-B1, we next tested whether platelets mechanistically potentiate endothelial cell dysfunction in SCD. HPAECs were co-cultured with platelets isolated from healthy vs steady-state SCD individuals. Platelets in co-culture were quiescent or activated with the agonists 0.1 U/mL thrombin, 25 ug/mL collagen, and 2.5 uM free heme for 6 hours, and HPAEC proliferation was measured via crystal violet staining 48 hours after exposure. We found HPAEC proliferation upon control and SCD platelet exposure to free heme, but SCD platelets exposed to heme induced a more robust response in HPAEC proliferation. In addition, we found that collagen only significantly promoted HPAEC proliferation in the presence of SCD platelets and not control platelets. Activated platelets, in the presence of hemolysis, release large amounts of TSP1 and TGF-B1 that may lead to enhanced endothelial cell proliferation and fibrosis in SCD, possibly via increased response to collagen by activated platelets. We will conduct further experiments to determine whether blockers of TSP-1 or its canonical CD47 endothelial receptor may abrogate aberrant cellular proliferation in SCD. Overall, the roles of TSP-1 and galectin-3 in pulmonary fibrotic injury via the TGF-B1 signaling pathway in SCD need to be further elucidated for therapeutic targeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

New Areas of Consensus for the Management of Sickle Cell Disease (SCD) in Pregnancy: An International Delphi Panel

Introduction and aim Sickle cell disease (SCD) is an autosomal recessive disorder characterized by a single nucleotide substitution in the beta-globin gene, resulting in the production of hemoglobin (Hb) S, and is the most common hematologic genetic disorder worldwide. As a result of Hb polymerization, oxidative stress and chronic hemolysis, complications occur during pregnancy for women with SCD, impacting both maternal and fetal health. We aimed to identify areas of consensus for the management of pregnancy-related complications in SCD, with a focus on prophylactic transfusion therapy (PTT). Methods A Delphi panel consisting of 13 experts from 4 countries replied to 2 rounds of questions (1 panelist dropped out between the 1st and 2nd survey). The 1st survey (October-November 2021) consisted of open-ended questions, while the 2nd one (April-May 2022, used to identify the degree of consensus or changes in opinion among panelists) was comprised of multiple-choice or closed questions based on the answers from the 1st survey. The thresholds were: <9/12 panelists (<75%) for no consensus, 9-10/12 panelists (75-90%) for consensus, and ≥11/12 panelists (>90%) for strong consensus. The Delphi panel covered topics where no global standardization or little evidence exists to guide management and prevention of maternal complications during pregnancy in SCD. These included antenatal care, hydroxyurea (HU) therapy, transfusion, prevention and treatment of complications, delivery and follow-up, bottlenecks and knowledge gaps. Results The panelists achieved strong consensus in the following areas of management: antenatal care (specifically, that pregnant women with SCD should be followed at least every 4 weeks by a multi-disciplinary team of specialists who would decide on the required tests and treatments), HU (discontinuation ≥3 months before conception; experts would not recommend discontinuation of pregnancy in case of exposure to HU during pregnancy), delivery and follow-up (that a multidisciplinary team should decide on labor induction or elective cesarean section). For the following areas of management, no consensus was achieved: continuation of HU therapy (could be considered during pregnancy/lactation if the risks of stopping HU therapy outweigh the benefits and if the patient understands the risks and agrees with continuing therapy), Hb level targeted prior to delivery, or preferred transfusion modality (simple transfusions versus automated red cell exchange [aRCX]) for a pregnant woman with SCD who was not on PTT during pregnancy but needed to be transfused prior to delivery. The input collected for transfusion (including details on when to recommend PTT) is summarized in the Table. The experts felt that target Hb and hematocrit should be set by clinical experience and would be impacted by the time interval between transfusions and the clinical indication for transfusion. There was no consensus on the most suitable transfusion modality. The experts identified a strong interest to prioritize the use of routine PTT in pregnant women with SCD as an area of research. Conclusion The experts did not achieve uniform consensus to support the standardized use of PTT in pregnant women with SCD. They achieved consensus that PTT should be routinely used in pregnant women with ≥2 acute vaso-occlusive pain episodes in a 4-week period regardless of genotype, with a preference for peripheral venous over central venous access, and the use of aRCX over simple transfusion (target HbS <30%) for the prevention of transfusional iron overload. The experts also achieved a strong consensus that prophylactic red cell exchanges should be used to reduce frequency of acute pain episodes in women taking high-dose opioids daily, and recommended consideration of on-demand transfusions for acute fetal complications. Consensus was not achieved on frequency of prophylactic transfusions, timing of PTT initiation during pregnancy in relation to HU discontinuation, target hematocrit levels, methods for the prevention of alloimmunization (extended red cell phenotyping versus genotyping) and the use of PTT for secondary prevention of fetal complications and pregnancy loss. In general, the areas of non-consensus seemed to correspond to areas with the widest knowledge gaps and lack of evidence, highlighting the need for further research in the use of PTT in pregnant women with SCD. Funding: Terumo Blood and Cell Technologies Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Balanced Crystalloids Versus Normal Saline in Noncritically Ill Adults with Sickle Cell Disease Admitted from the Emergency Department: A Sub-Analysis of the Salt-ED Trial

Background: Common acute manifestations of sickle cell disease (SCD), including vaso-occlusive pain episodes (VOE) and other complications, are often treated in the emergency department (ED). Effective treatment in the ED remains challenging and often only includes supportive care, such as analgesics and intravenous (IV) fluids. There is clinical equipoise regarding the type of IV fluid to use for resuscitation, and while normal saline (NS) is the most commonly one used in the ED, some SCD providers discourage use of NS. Patients with SCD and kidney dysfunction, who are often at-risk for acidosis already, may not effectively excrete the high sodium, hyperosmolar load of NS, and in large volumes NS may cause hyperchloremic metabolic acidosis that could impact sickle red blood cell (sRBC) rheology (Nath, Nat Rev Nephrol, 2015). We recently demonstrated modestly hyperosmolar and acidotic fluids like NS (308mOsm/L, pH=5.5) may dehydrate sRBCs, increase sRBC/endothelium adhesion, and worsen deformability; and we also showed NS bolus use in pediatric pain management in the ED was associated with inferior pain control, increased likelihood of hospital admission, and more time spent in the ED (Carden et al., Blood & AJH, 2017&2019). Rigorous studies examining the impact of IV fluids, particularly NS, during ED management of adults with SCD have not been performed, and recent evidence suggests that balanced crystalloids (B) may be safer for resuscitation of adults in the ED. Methods: We utilized data from the seminal SALT-ED study, a single-center, pragmatic, multiple-crossover prospectively randomized controlled trial (RCT) that compared outcomes related to administration of balanced crystalloids (primarily lactated ringers [LR], 274mOsm/L, pH=6.5) to NS administration to all-comer adult patients in the ED (Self et al., NEJM, 2018). Using ICD-10 codes, we conducted an intention-to-treat sub-analysis of SCD patients in the SALT-ED trial. Enrolled participants were non-critically ill adults (18 years and older) with SCD who presented to the ED and were subsequently hospitalized to a non-ICU bed at the hospital. Chief complaint was captured when available. The primary outcome examined was median hospital free days post-index hospitalization and secondary outcomes included a composite of adverse kidney events and death (i.e. MAKE30), as well as changes in serum electrolyte status. Results: There were 150 adults with SCD identified in the SALT-ED study (Table 1a). Of 13,347 visits captured, 247 ED visits were attributed to these 150 patients - N=126 visits (51%) in the B group and N=121 (49%) in the NS group. Over 50% of documented chief complaints in the ED were pain-related. Compared to all adult patients in the SALT-ED study, those with SCD were younger, more likely to identify as female and Black, have a lower baseline creatinine, and while having similar hospital free days and similar volumes of crystalloids administered in the ED, patients with SCD had an approximate 3.5 times higher odds of suffering an acute kidney injury event (i.e. MAKE30 endpoint) during hospitalization. Compared to those with SCD randomized to the B group, those patients with SCD in the NS group had more adverse kidney events, increased mean length of hospital stay, higher maximum in-hospital sodium, higher maximum in-hospital chloride, and lower minimum in-hospital bicarbonate, suggesting an increased risk of high sodium acidosis with NS use in the ED (Table 1a&b). Using combined data from both groups and analyzing with linear regression, we found in-hospital electrolyte levels were significantly associated with hospital length of stay among adults with SCD administered IV fluids in the ED (Table 1c). Conclusion: The results from this large RCT sub-analysis further support recent evidence suggesting NS is not an ideal resuscitation IV fluid for adults with SCD presenting to the ED with pain and other complications. Adult patients with SCD randomized to NS in the ED had higher rates of in-hospital high sodium levels, metabolic acidosis, increased hospital stay, and increased kidney events. Our findings suggest that administering balanced crystalloids like LR may be a better option for IV fluid resuscitation among adults with SCD presenting to the ED. Further research is needed to confirm these findings and to determine the optimal IV fluid regimen for adults with SCD presenting to the ED with VOE or other complications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Assessing the Recovery Process after a Vaso Occlusive Crisis

Introduction: Adults with sickle cell disease (SCD) suffer from vaso-occlusive pain episodes (VOEs) for which they often receive care in infusion clinics (ICs). Typically, 80% of patients treated for VOE are discharged home but it is not clear how long it takes to recover to baseline functioning after an acute visit.1 Recent clinical trials have struggled to identify practical and useful outcomes for novel therapies that decrease the length of VOE or pain during hospitalization. As many patients with VOE are seen and treated in an IC and discharged home, identifying potential endpoints for novel therapies in this site of care are needed. The purpose of this study was to measure time to recovery for two weeks post an IC visit for management of VOE and to identify patient characteristics that predict a return visit for pain management within that time period. Methods: A convenience sample of adults with SCD (≥18YO) were consented to and enrolled into this IRB-approved study during a visit to the IC for VOE. Medical records of all subjects were reviewed to collect data on the number of acute visits, hospitalizations, use of disease-modifying therapies, use of pain medications, baseline laboratory data, and comorbidities. Data was collected on the date VOE pain started, pain scores before and after treatment, and analgesic management. Baseline surveys were collected on the day of the visit and patients were sent follow-up surveys after discharge electronically through REDCap on post-discharge days 2,4,6,8,10,12,14. Participants were asked to rate their pain on a 0-10 Visual Analog Scale. The PROMIS pain interference and Fatigue scales were used to assess pain impact and the Subjective Opioid Withdrawal Scale (SOWS) was used to assess for signs of opioid withdrawal. Return to baseline functioning was estimated by asking participants at each contact post-visit to rate their current level of functioning in activities of daily living on a scale of 0-100, with 100 being their baseline level of function. A similar scale was used to measure the resolution of pain, with the anchor being "back to your usual, non-crisis, level of daily pain." Subjects were encouraged to complete surveys through text message reminders and were compensated with a fifty-dollar gift card if all surveys were completed. Cox proportional hazards modeling was used to examine predictors of time to a return acute care visit within 14 days of the index visit. Results: Table 1 shows the characteristics of the 48 patients enrolled in the study. Despite multiple text/email reminders, few patients completed all the surveys (8/48). Thirty-two out of 48 people completed initial surveys. Of the 17 patients who completed a day 2 or later SOWS score 7 had moderate or severe withdrawal symptoms and of those 3 had a return visit within 14 days while 5/10 of those with mild scores had a return visit. Nineteen out of 48 patients returned for treatment within 14 days. Controlling for pain at discharge, sex, and genotype only the number of acute visits in the six months prior to the index visit was associated with a return visit in 14 days (HR 1.31, 95% CI: 1.13 1.53). The figure shows the probability of a return visit over time by genotype. The median number of visits the entire cohort had in the six months prior to the index visit was 4.5 (IQR: 2,10) however, in those patients that returned for treatment within 14 days the median number of visits in the last six months was 8 (IQR: 5,11). Conclusions: In this study of patients' experience post an acute visit for pain management at an IC, we identified that the number of acute visits in the six months prior to the index visit predicted a return visit within 14 days. It was challenging for patients to complete surveys after their acute visits suggesting that this was not a feasible way to collect these important patient-reported outcomes. With almost 40% of patients returning for an acute care visit within 14 days, decreasing the percentage of patients with return visits may be a potential outcome for trials of novel therapies to treat VOE in the IC setting. 1. Lanzkron S, Carroll CP, Hill P, David M, Paul N, Haywood C, Jr. Impact of a dedicated infusion clinic for acute management of adults with sickle cell pain crisis. Am J Hematol. 2015;90(5):376-380. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ketamine Use for Management of Vaso-Occlusive Pain in Children with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) vaso-occlusive pain episode (VOE) management often includes non-steroidal anti-inflammatory drugs (NSAIDs) and opioids, which can be inadequate and associated with significant side effects. Ketamine, a dissociative anesthetic, produces potent analgesic effects by blocking N-methyl-D-aspartate (NMDA) receptors, which impairs sensitization of spinal neurons to nociceptive stimuli. This mechanism allows ketamine to mitigate neuropathic pain and modulate opioid tolerance and opioid-induced hyperalgesias. Although only FDA-approved for use as an anesthetic agent, limited data suggest that low-dose ketamine is a safe and potentially effective adjunctive treatment for VOE pain. Objectives: This study aimed to characterize ketamine use for VOE management in children with SCD. Method: This retrospective cohort study summarizes a single-center experience regarding the use of ketamine for inpatient management of VOE in 156 admissions from 2014 to 2020 in which 44 unique children received ketamine. Results: Continuous low-dose ketamine infusion was most commonly prescribed to adolescents as an adjunct to opioids (median starting dose 2.0 mcg/kg/min; median maximum dose 3.0 mcg/kg/min). Ketamine was started a median of 13.7 hours after admission. Median ketamine infusion duration was 3 days (IQR 1.8-4.3). For admissions in which opioid was decreased during ketamine infusion, the median time from start of ketamine infusion to first opioid dose adjustment was 12.5 hours (IQR 3.25-36.6). In most encounters (n=119, 76.3%), ketamine infusion was discontinued prior to opioid PCA discontinuation. In 33 (21.2%) of encounters, the ketamine dose was decreased within 6 hours prior to discontinuation. The majority of encounters (79.3%) had a reduction in either PCA dose, continuous opioid infusion, or both while receiving ketamine. Low-dose ketamine infusion was well-tolerated overall, with side effects noted in 21.8% (n=34) of encounters. The most common side effects included dizziness (5.6%), hallucinations (5.1%), dissociation (2.6%), and sedation (1.9%). Nearly all side effects were reversible, resolving within 24 hours of dose reduction (n=11, 78.6%) or infusion discontinuation (n=21, 95.5%). There was no significant difference in patient sex or age at admission between groups with reported side effects compared to those without. There was a significant difference in hemoglobin genotype between groups (p=0.005). In both groups, most patients had hemoglobin SS. However, a higher percentage of encounters with side effects included patients with hemoglobin SC. Further, a higher percentage of patients in the no side effect group received hydroxyurea before admission (p=0.032) (Table 2). There were no reports of ketamine withdrawal. In 11 (7.1%) encounters, benzodiazepines were administered during ketamine infusion; 7 intravenously and 4 enterally. In most cases (n=5, 45.5%) the benzodiazepine was given for anxiety, in 2 cases (18.2%) for spasticity, 2 (18.2%) for spasm, 1 (9.1%) for agitation and 1 (9.1%) for pain. Benzodiazepines were administered within 12 hours after ketamine initiation in only 3 cases. Most patients who received ketamine went on to receive it again during a subsequent admission (72.1%, n=98). Conclusion: Ketamine infusion should be considered when developing individualized pain plans for children with SCD, and it may be particularly beneficial for those who have a history of inadequate pain relief from opioids or dose-limiting opioid side effects. The variability of ketamine administration highlights the need for standardized protocols for ketamine use in VOE management. Further prospective study is needed to establish the efficacy of ketamine infusion and to determine optimal dosing, which may be patient-specific. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Urinary C-Telopeptide of Type II Collagen As a Marker of Avascular Necrosis in Younger and Older Adults with Sickle Cell Disease

Background Avascular necrosis (AVN) is a common cause of morbidity and impaired mobility in adults with sickle cell disease (SCD). Repeated episodes of vaso-occlusion cause bone infarctions that lead to AVN. The early detection of AVN in adults with SCD requires expensive imaging modalities (CT, MRI) as it is often not detected on plain radiographs until advanced stages. Type II collagen is an abundant protein of the cartilage matrix and its degradation product, urinary type II collagen C-telopeptide (UCTX II) is a specific biomarker used to measure degradation of articular cartilage in adults with osteoarthritis (OA). UCTX II is easily measured in urine samples and may facilitate early detection of AVN and other bone diseases seen in SCD. The primary objective of this study is to assess the relationship between concentrations of UCTX II and avascular necrosis in adults with SCD. Methods We included adults (≥ 18 years) with a laboratory confirmed diagnosis of SCD in this cross-sectional study. We collected sociodemographic data by patient survey and extracted medical history and routine laboratory data from the electronic health record including history of AVN and joint surgery which were confirmed by participant survey. History of arthritis included in the participant survey. We collected urine and blood specimens and administered physical performance measures on the same day during the participant's usual state of health. We defined usual health as no hospitalizations in the last 6 weeks and no emergency room or Sickle Cell Day Hospital visits in the 2 weeks prior to the assessment and specimen collection. We quantified UCTX II by ELISA and corrected for creatinine excretion. We used descriptive statistics and visual displays to summarize the data and compared UCTX II levels between participants with and without AVN using Wilcoxon rank sum test. We evaluated relationships between UCTX II and physical performance measures using Spearman's correlation coefficients. Results We measured UCTX II in 34 participants. The median age of participants was 48.5 years (range 20-71). Most (62%) participants were ≥ 40 and 38% were between 18-39 years old. SCD genotypes included HbSS (56%), HbSβ0 (3%) and HbSβ+-thalassemia (12%), and HbSC (29%). Most participants (59%) had a diagnosis of AVN, 24% had a history of joint surgery, and 29% had reported a history of arthritis. The median UCTX II level was 348 ng/mmol creatinine (IQR 229-534; range 89 - 78230). Younger adults had significantly higher median UCTX II levels compared to older adults with a median UCTX II of 536 (IQR 358 - 999) in young adults versus 242 (IQR 180 - 357) in older adults; p-value = 0.001. There was not a statistically significant difference in median UCTX II level between participants with and without AVN, history of joint surgery, and self-reported history of arthritis. When we controlled for age using a rank-based nonparametric ANCOVA, there remained no difference in UCTX II levels between participants with and without AVN (p-value = 0.68). Participants with a history of AVN had a median UCTX II level of 311 (IQR 209 - 426) compared to a median of 384 (IQR 248 - 704) in participants without AVN; p-value = 0.22. Participants with a history of joint surgery had a median UCTX II level of 283 (IQR 139 - 371) compared to participants with no history of joint surgery with a median of 357 (IQR 233 - 620); p-value = 0.27. Participants with a history of self-reported arthritis had a median UCTX II level of 312 (IQR 245 - 353) compared to patients without arthritis who had a median of UCTX II of 382 (IQR 217 - 673); p-value = 0.27. Conclusion This preliminary data showed no significant difference in UCTX II levels between adults with and without AVN, history of joint surgery, or self-reported history of arthritis; however, the small sample size limited statistical power to detect differences. We found that younger adults with SCD had higher UCTX II levels than older adults with SCD. The increased UTCXII in younger adults with SCD is consistent with prior studies that demonstrate higher UCTXII in young adults and a gradual decline with age due growth plate activity that contributes to the higher UCTXII levels seen in young adults. The role of UCTXII will need to be explored further in a larger sample and compared to radiographic staging of AVN that includes evaluation of joint damage. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intravenous Fluid Replacement Therapy and Hospital Outcomes for Vaso-Occlusive Episodes in Patients with Sickle Cell Disease

Background: Vaso-occlusive event (VOE) is the most common reason for hospitalization in pediatric patients with sickle cell disease (SCD) and can cause significant morbidity and mortality. Fluid replacement in patients with VOE can reduce the sickling process and volume replete, however clinical practices regarding fluid therapy in SCD vary across different institutions. A large contributor to this variation is the lack of evidence surrounding the amount, rate, or type of fluid that should be used during a pain crisis episode. The goal of this retrospective analysis is to examine the relationship of average amount of fluid replacement with length of hospitalization and risk for adverse events, such as acute chest syndrome (ACS), transfer to the pediatric intensive care unit (ICU) and readmission (within 4 weeks from previous admission), at a large academic institution. Our hypothesis is that excessive fluid therapy prolongs hospital length of stay (HLOS) and increases risk for the aforementioned adverse events. Methods: This was a retrospective analysis from a single center between January 2015 and April 2020. Patients with SCD (HbSS, HbSC, HbS-β0 thalassemia, or HbS-β⁺ thalassemia), aged 0- 30 years, with consecutive admissions hospitalized for VOE were included after local IRB approval. Records were reviewed for demographic variables such as age and gender, as well as clinical parameters, such as intravenous fluid therapy, tonicity, total fluid amount, medical history, intravenous pain medication, oral pain medication, and treatment during hospitalization. Results: A total of 617 hospitalizations in 162 patients were included in this study. One hundred forty-one (22.9%) of the 617 hospitalizations experienced ACS; and in 55 (9%) of these hospitalizations, ACS developed during the hospital stay. Fifteen (2.4%) of the 617 hospitalizations required transfer to the pediatric ICU; 7 of the 15 hospitalizations required transfer for exchange transfusion, 7 required transfer for acute hypoxemic respiratory failure, and 1 for hypertension. One hundred twenty-one (19.7%) of the 617 hospitalizations required readmission within four weeks of their previous admission. A Pearson correlation coefficient assessing the relationship between HLOS (days) and average IV fluid over admission (ml/kg/day) showed that more IV fluid therapy was associated with shorter HLOS (r = -0.140 and p = < 0.01). In a univariate analysis, oxygen requirement, acute chest syndrome, ICU transfer, readmission within four weeks, history of chronic pain, treatment with hydroxyurea, not receiving a normal saline bolus, and requiring a transfusion were associated with a prolonged hospital stay (p = < 0.05). A multivariable linear regression showed that ICU transfer, readmission within four weeks, history of chronic pain, treatment with hydroxyurea and requiring a transfusion were significantly associated with a prolonged hospital stay. Moreover, oxygen requirement and requiring a transfusion were significantly associated with developing ACS during their hospitalization (OR=11.094, 95% CI=5.32, 23.15) and (OR=4.69, 95% CI= 2.18, 10.09), respectively. Chronic pain was significantly associated with readmission within four weeks (OR=6.40, 95% CI=3.84, 10.65). Conclusions: This retrospective study suggests that IV fluid therapy was associated with shorter HLOS and lower incidence of complications such as ACS. However, clinical parameters such as chronic pain and treatment with hydroxyurea were associated with prolonged LOS. These results differ from previous studies and highlight the need for prospective multicenter studies to clarify the relationship between IV fluid therapy, HLOS and adverse events in patients with sickle cell disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Heme and Iron Aggravate Tissue Damage in Sickle Cell Disease By Driving Macrophage Functional Reprogramming through the Coordinated Suppression of Efferocytosis and Mitochondrial Metabolism

Introduction: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is in part driven by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme released upon hemolysis, which acts as damage-associated molecular pattern to stimulate macrophage inflammatory phenotypic switching through TLR4 signaling and ROS production (Vinchi et al., Blood 2016). Heme-induced pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to the inflammatory landscape and organ damage typically associated with this disease. While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties remains unexplored. Macrophage functions such as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages (BMDMs), to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme severely alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme excess drives defective efferocytosis of apoptotic neutrophils in BMDMs and mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the nuclear transcription factor PPARγ and its coactivator PGC1α, which lead to reduced expression of efferocytic receptors and impaired mitochondrial dynamics and functions. This results in limited recognition/engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4 and IL-10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. Heme-driven changes are associated with cell metabolic skewing towards glycolysis and pentose phosphate pathway and reduced reliance on mitochondrial fatty acid oxidation and TCA cycle. Macrophages pre-loaded with fatty acids showed decreased oxygen consumption rate and ATP production after heme exposure, indicating that heme-altered mitochondrial dynamics impair lipid catabolism via limited mitochondrial oxidative phosphorylation. Overall, heme induces macrophage phenotypic and functional reprogramming through the coordinated suppression of efferocytosis, via efferocytosis receptor downregulation, and fatty acid β-oxidation, via altered mitochondrial dynamics. We further demonstrate that heme-impaired efferocytosis and tissue damage promote autoimmunity through anti-nuclear antibody production against 'self' secondary necrotic cells, potentially explaining the increasing evidence of autoimmune diseases in SCD patients. Finally, hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment improve tissue damage in hemolytic mice through macrophage rewiring. Defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients' plasma and rescued by heme scavenging via haptoglobin/hemopexin and PPARγ/PGC1α modulation via PPARα agonist or IL-4. Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation significantly promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events and prevent autoimmune conditions.

Baseline Patient and Provider Reports of Quality of Care and Confidence Providing ED Management of Vaso-Occlusive Episodes

Baseline Patient and Provider Reports of Quality of Care and Confidence Providing ED Management of Vaso-Occlusive Episodes

Dietary iron restriction protects against vaso-occlusion and organ damage in murine sickle cell disease

AbstractSickle cell disease (SCD) is an inherited disorder resulting from a β-globin gene mutation, and SCD patients experience erythrocyte sickling, vaso-occlusive episodes (VOE), and progressive organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron overload, and another subpopulation of SCD patients manifest iron deficiency. To elucidate connections between dietary iron, the microbiome, and SCD pathogenesis, we treated SCD mice with an iron-restricted diet (IRD). IRD treatment reduced iron availability and hemolysis, decreased acute VOE, and ameliorated chronic organ damage in SCD mice. Our results extend previous studies indicating that the gut microbiota regulate disease in SCD mice. IRD alters microbiota load and improves gut integrity, together preventing crosstalk between the gut microbiome and inflammatory factors such as aged neutrophils, dampening VOE, and organ damage. These findings provide strong evidence for the therapeutic potential of manipulating iron homeostasis and the gut microbiome to ameliorate SCD pathophysiology. Many treatments, which are under development, focus on lowering the systemic iron concentration to relieve disease complications, and our data suggest that iron-induced changes in microbiota load and gut integrity are related- and novel-therapeutic targets.

PSAT066 Adrenal Insufficiency Associated with Amyloidosis and Sickle Cell Disease

Abstract Background Amyloidosis causes chronic inflammation by the deposition of amyloid protein fragments in multiple organs, such as kidneys, adrenal, and pituitary glands. It is a challenge to access adrenocortical function in renal amyloidosis patients due to heavy proteinuria induced hypoproteinemia and low corticosteroid-binding globulin (CBG) which may cause false positive adrenocorticotropin (ACTH) test and the reduced cortisol level uncorrelated with free cortisol. On the other hand, adrenal insufficiency also frequently occurs in Sickle Cell Disease (SCD) patients. The hypothalamic-pituitary-adrenal (HPA) axis can be affected by tissue hypoxia during recurrent small vessel occlusion, chronic anemia, and iron-overload related oxidative stress. We report a SCD patient with nephrotic syndrome due to amyloidosis, who has adrenal insufficiency. Case Presentation A 41 years old female with Nephrotic syndrome secondary to Amyloid A (AA) amyloidosis, Adrenal insufficiency, Sickle cell disease (HbSC), Graves’ Hyperthyroidism, CVA, and bilateral femoral head avascular necrosis, presented with sickle cell pain crisis and anasarca with hypotension. She was on Hydrocortisone for 12 years but was not compliant for 2 months. She was on Methimazole for several years before it was discontinued one year ago. She has amenorrhea, severe edema with anasarca from the lower extremities to the abdomen. Labs showed morning cortisol 5.46 ug/dL, low albumin 2.4 g/dL, high TSH 5.43 uIU/mL, normal FT4 1.60 ng/dL, high urine protein 996.2 mg/dL, and eGFR 21.2. CT revealed pericardial effusion, free pelvic fluid, splenomegaly, edema, and thyroid goiter. Previous labs showed persistent low cortisol 0.9 to 3.5 ug/dL, high ferritin 1007 ng/mL, and pituitary workup was normal for Prolactin 4.6 ug/L, LH 5.02 mIU/mL, FSH 9.9 mIU/mL, IGF1 216 ng/mL, and ACTH 13.1 pg/mL. NM thyroid uptake elevated 51.7% and positive TPO 79.7 IU/mL. CT head was unremarkable. The patient was managed with Lasix and Albumin infusion for anasarca, Cyclosporine for amyloidosis, and Midodrine 10 mg TID for hypotension. Hydrocortisone stress dose was given followed by Prednisone 30 mg BID. Her symptoms improved. Discussion In Amyloidosis patients with heavy proteinuria, the low serum CBG levels affect total cortisol level. Therefore, the proper assessment for the adrenal function should be used with albumin-calculated free cortisol or cortisol/CBG ratio in addition to the ACTH stimulation tests. It is essential to differentiate between adrenal insufficiency and amyloidosis-related symptoms to prevent adrenal crisis and refrain from glucocorticoid side effects by unnecessary steroid use. On the other hand, endocrine manifestations of SCD may include hypogonadism, thyroid, adrenal, and pituitary dysfunction due to vaso-occlusive episodes induced multi-organ ischemic damage. This patient has amenorrhea, adrenal insufficiency, and normalized thyroid function test in Graves’ disease. It is crucial to avoid iron-overload induced oxidative stress which would worsen endocrine dysfunctions as well. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.