Hypoxia Responsiveness in RBCs from Patients with Sickle Cell Disease Associates with a More Severe Clinical Phenotype

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3643-3643 ◽  
Author(s):  
Myeongseop Kim ◽  
Yunus Alapan ◽  
Anima Adhikari ◽  
Jane A. Little ◽  
Umut A. Gurkan
Keyword(s):  
Sickle Cell Disease ◽  
Oxygen Tension ◽  
Sickle Cell ◽  
Patient Population ◽  
Cellular Interactions ◽  
Cell Disease ◽  
Flow Conditions ◽  
Hypoxic Conditions ◽  
Clinical Associations ◽  
One Way Anova

Abstract Abnormal cellular interactions and adhesion of blood cells to endothelium under hypoxic conditions are pathophysiologically central to Sickle Cell Disease (SCD). Analysis of the germane cellular interactions in physiologically relevant flow and oxygen tension conditions is technically challenging. We developed a low-cost, single-use micro-gas exchanger, integrated with a microfluidic device, with which to evaluate the effects of hypoxia. This system can simulate a range of physiological conditions, under which to analyze red blood cell (RBC) adhesion to endothelial or sub-endothelial components (here fibronectin, FN, or laminin, LN). 15 µL of surplus whole blood was injected on to FN- or LN-functionalized microchannels, after passing through a micro-gas exchanger composed of a gas permeable inner tubing inside a gas impermeable outer tubing (Fig. 1A). Blood flowed, and was deoxygenated, within the inner gas permeable tubing, while a controlled gas mixture (5% CO2 and 95% N2) flowed in the space between inner and outer tubing. Blood samples were flown into microchannels at physiological flow conditions (1 dyne/cm2), and at normoxic (98% SpO2) or hypoxic oxygen saturations (83% SpO2). Serial quantitative evaluation of RBC adhesion, using standardized protocols, were on >35 subjects (13 males and 22 females) with homozygous HbSS patients on whom we have correlative clinical data (institutional review board approved study). Significant heterogeneity in the RBC adherence response to hypoxia was seen among SCD patients. RBCs from a hypoxia responsive (HR) population showed a significantly greater increase in adhesion compared to RBCs from a hypoxia non-responsive (non-HR) population, for both FN and LN (Fig. 1B, p=0.0001, one-way ANOVA). RBCs from the non-HR patient population had higher HbF values than did RBCs from the HR patient population (p=0.0079, one-way ANOVA). All HR RBCs had <7% HbF. Furthermore, patients with HR RBCs had significantly higher serum ferritin (p=0.0024, one-way ANOVA) and LDH levels (p=0.0128, one-way ANOVA), and higher reticulocyte counts (Fig. 1C, p<0.0001, one-way ANOVA) than did patients with non-HR RBCs. We also observed that 7 out of 8 subjects in HR subpopulation were transfused, whereas only 2 out of 15 subjects in the non-HR subpopulation were transfused (Fig. 1C). Moreover, the age distribution of HR patients was significantly younger (<35 years) than non-HR patients (Fig. 1D, p<0.05, Chi-Square). We suspect that the HR RBC phenotype associates with greater clinical disease activity, and more intensive therapeutic interventions, i.e., transfusions, overall. Here, we reported adhesion of RBCs to endothelium associated proteins, FN and LN, under normoxic and hypoxic conditions, in a closed microscale environment under physiologically relevant flow conditions. The approach and technique presented here enabled us to control the oxygen tension of the blood in flow and to quantify the adhesion response of RBCs with SCD in a cost-efficient and patient-specific manner, demonstrating the effectiveness of our system. We identified a unique patient population whose RBCs, designated as 'hypoxia-responsive' (HR), showed enhanced adhesion, to LN and FN, in response to hypoxia in vitro. Clinical correlates suggest a more severe clinical in vivo SCD phenotype in this subgroup. Acknowledgments: This work was supported by Grant # 2013126 from the Doris Duke Charitable Foundation and National Heart Lung and Blood Institute R01HL133574. Authors acknowledge Grace Gongaware for scientific illustrations, and contributions of patients and health care providers at University Hospitals. Figure Interrogation of RBC hypoxia responsiveness and clinical associations in SCD. (A) Blood deoxygenates at the micro-gas exchanger during flow. (B) Clustered patients in HR and non-HR populations, based on increase in RBC adhesion in response to hypoxia. HR patients showed significantly greater increase in the number of adhered RBCs with hypoxia compared to non-HR patients (p<0.05). (C) Patients with HR RBCs showed significantly greater reticulocyte counts compared to patients with non-HR RBCs. 7 out of 8 subjects in the HR patient subpopulation were transfused (>10% Hb A); 2 out of 15 subjects in the non-HR subpopulation were transfused. (D) Hypoxia responsive patients (N=8) were younger (<35 years). 8/16 subjects <35 years old and 0/10 subjects ≥35 years old were HR (p<0.05). Figure. Interrogation of RBC hypoxia responsiveness and clinical associations in SCD. (A) Blood deoxygenates at the micro-gas exchanger during flow. (B) Clustered patients in HR and non-HR populations, based on increase in RBC adhesion in response to hypoxia. HR patients showed significantly greater increase in the number of adhered RBCs with hypoxia compared to non-HR patients (p<0.05). (C) Patients with HR RBCs showed significantly greater reticulocyte counts compared to patients with non-HR RBCs. 7 out of 8 subjects in the HR patient subpopulation were transfused (>10% Hb A); 2 out of 15 subjects in the non-HR subpopulation were transfused. (D) Hypoxia responsive patients (N=8) were younger (<35 years). 8/16 subjects <35 years old and 0/10 subjects ≥35 years old were HR (p<0.05). Disclosures No relevant conflicts of interest to declare.

scholarly journals CD209-336A/G promotor polymorphism and its clinical associations in sickle cell disease Egyptian Pediatric patients

2018 ◽  
Vol 11 (2) ◽  
pp. 75-81 ◽  
Author(s):  
Rasha Abdel-Raouf Afifi ◽  
Dina Kamal ◽  
Riham El. Sayed ◽  
Sherif M.M. Ekladious ◽  
Gehan H. Shaheen ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Pediatric Patients ◽  
Cell Disease ◽  
Clinical Associations

Vasoocclusive Painful Episodes and the Risk of Acute Chest Syndrome in Children with Sickle Cell Disease: Does the Selection of Parenteral Opioid Matter?

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 442-442
Author(s):  
Ram V. Kalpatthi ◽  
Matt Hall ◽  
Jignesh Dalal ◽  
Gerald M Woods
Keyword(s):  
Sickle Cell Disease ◽  
Length Of Stay ◽  
Respiratory Depression ◽  
Sickle Cell ◽  
Patient Population ◽  
Lower Risk ◽  
Acute Chest Syndrome ◽  
Cell Disease ◽  
Median Length ◽  
Hospitalization Costs

Abstract Background: Vaso-occlusive painful episodes (VOC) is the most common cause of hospitalization in patients with sickle cell disease (SCD) that often requires opioid analgesics and intravenous fluids. However, due to respiratory depression, the use of opioids during these painful episodes has been associated with the risk of development of acute chest syndrome (ACS), the major cause of early mortality in these patients. Nalbuphine is a unique opioid analgesic that effectively treats pain but lacks significant respiratory depression. Previous small scale studies (Pediatr Blood Cancer 2005 and J Pediatr Hematol Oncol 2011) suggested a lower risk of ACS in patients receiving Nalbuphine during VOC when compared with morphine. We sought to compare outcomes and the risk of ACS in pediatric sickle cell patients hospitalized for VOC in relationship to different opioid treatments. Methods: We used the Pediatric Health Information System (PHIS), an administrative database of children's hospitals in the US. Patients ≤ 21 years of age with SCD from 43 hospitals from 2005-2013 were included in the study. SCD patients who had a primary diagnosis VOC (ICD-9 codes 282.62, 282.64, 282.42 and 282.69) were included. VOC hospitalizations were divided into four different narcotic analgesic groups. In bivariate analyses, we compared patient demographics, treatment details, risk of ACS, length of stay, hospital costs, and readmissions across groups using chi-square tests or Kruskal-Wallis tests as appropriate. Multivariable models accounted for hospital clustering with generalized estimating equations for binary outcomes and generalized linear mixed effects models with random hospital intercepts and an exponential distribution (due to skewness of the data) for continuous outcomes. A p-value <.05 was considered statistically significant. Results: From 2005 to 2013, a total of 11260 unique pediatric SCD patients were identified. These patients had 42688 VOC hospitalizations, and received a single parenteral narcotic analgesic during each hospitalization (Morphine 82.2%, Hydromorphone 13.3%, Fentanyl 2.4% and Nalbuphine 2.1% of VOC hospitalizations). Table 1 describes the outcomes of VOC hospitalizations for all four narcotic groups. In unadjusted analysis, patients who received Nalbuphine only had significantly lower risk of ACS diagnosis (5.6%), ICU admissions (0.9%), shorter median length of stay (2 days) and lower median hospitalization costs ($4345) [Table 1]. Patients who received Morphine had less readmission within 3 and 7 days of discharge after VOC hospitalizations (Table 1). Multivariate analysis after adjusting for age, gender, hospital, season, race, payer source, and complex chronic conditions confirmed that patients who received Nalbuphine had significantly lower risk of ACS diagnosis, ICU admissions, shorter median length of stay, and lower median hospitalization costs compared to patients who received Morphine, Hydromorphone or Fentanyl (Table 2). Conclusions: In our largest pediatric in-patient sickle cell cohort, Nalbuphine was associated with significantly lower risk of development of ACS when compared with other opioids. In addition, patients received Nalbuphine had significantly lower ICU admissions, shorter hospital stay and lower hospitalization costs suggesting better pain control. However, our study shows that Nalbuphine in this patient population is rarely utilized. Prospective studies are needed to confirm this association and to elucidate the mechanisms that underlie these beneficial effects of Nalbuphine in this patient population. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Complication Rates in Patients with Sickle Cell Disease Living Chronically at Moderate Altitude

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4095-4095 ◽  
Author(s):  
Claire L Stokes ◽  
Christopher M McKinney ◽  
Christopher C Silliman
Keyword(s):  
Sickle Cell Disease ◽  
Sea Level ◽  
Oxygen Tension ◽  
Sickle Cell ◽  
Rate Ratio ◽  
Incidence Rates ◽  
Complication Rates ◽  
Moderate Altitude ◽  
Cell Disease ◽  
Splenic Sequestration

Abstract Background: The increased risk of acute vaso-occlusive pain crisis and splenic infarction in children with sickle cell disease (SCD) acutely exposed to altitude has been well documented. However, little is known about complication rates in children chronically living at moderate altitude. We hypothesize that children with SCD experience more complications than children with SCD living at sea level. Methods: A retrospective chart review of all patients with sickle cell disease followed at the Children's Hospital Colorado between January 2001 and December 2010 was completed. Patients observed for less than one year were excluded from analysis. Incidence rates for vaso-occlusive crisis (VOC), acute chest syndrome (ACS), splenic sequestration, and stroke were calculated. Rate ratios and 95% confidence intervals were determined comparing to children with SCD from institutions located near sea level. Secondary measures looked at baseline hematologic indices collected at annual comprehensive care visits and percentage of patients with abnormal tricuspid regurgitation jet velocity (TRV). Two-tailed Student's t-tests were used to compare means of continuous variables. Results: 179 children were observed for a total of 1032.37 patient-years with demographics (Table 1). . At moderate altitude, patients with Hgb SS experienced about a 20% higher rate of VOC compared to historic controls with a rate ratio of 1.19 (1.03-1.39) (Table 2). Patients with Hgb SC had almost 3 times the number of admissions for splenic sequestration than those at sea level with a rate ratio of 2.93 (1.05-8.02). Incidence rates for ACS and stroke appeared to have been higher at moderate altitude than sea level, but did not reach statistical significance. There was also no difference in the percentage of patients with abnormal TRV. Baseline lab values were less than the 95%ile except for the hemoglobin of 11.7 for SC patients (Brown et al 1994). Discussion: The oxygen tension at an elevation of 5,280 feet (1,609 m) is 20% lower than at sea level due to the reduction in barometric pressure. Reduced oxygen tension may lead to increased hemoglobin S polymerization and red cell sickling. Hemoglobin SC patients have higher baseline hemoglobins, and their increase in splenic sequestration may be due to increased blood viscosity. Interestingly, the rate of ACS and pulmonary hypertension did not seem to be significantly elevated in our patients living at moderate altitude. This may be due to a lack of statistical power given the small size of this single institution study. Another limitation of this study is the comparison to data from multiple institutions near sea level which does not necessarily control for other possible contributing factors, e.g. climate. Also, VOC events were defined as hospitalizations requiring parenteral opioid administration, which is a stricter definition than used in the sea-level data. Thus, the risk ratio may be underestimated. Nevertheless, the data supports the anecdotal experience that patients living chronically at moderate altitude have increased sickle cell-related complication rates. Table 1 Table 1. Demographics Table 2 Table 2. Complication Incidence Rates 1 -compared to Gill et. al, Blood, 1995 2 - compared to Vichinsky et. al, Blood, 2012 3- compared to Brousse et. al, BJH, 1997 4- compared to Pashankar et. al, Pediatrics, 2007 5- compared to Quinn et. al, Blood, 2008 Disclosures No relevant conflicts of interest to declare.

Platelet Nucleation on Arrested Neutrophils Drives Vaso-Occlusion in Sickle Cell Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 414-414
Author(s):  
Maritza A. Jimenez ◽  
Prithu Sundd ◽  
Enrico M Novelli ◽  
Gregory J Kato
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Fluorescence Microscopy ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Genetic Disorder ◽  
Epifluorescence Microscopy ◽  
Cellular Interactions ◽  
Cell Disease ◽  
Neutrophil Aggregation

Abstract Introduction: Sickle Cell Disease (SCD) is an autosomal recessive genetic disorder that leads to sickling and hemolysis of RBCs under hypoxic conditions. As a result of chronic hemolysis, SCD is associated with a hyper-inflammatory and hyper-coagulation state, which accounts for enhanced adhesion of leukocytes, platelets, RBCs and vascular endothelial cells leading to vaso-occlusion. Acute vaso-occlusive pain crisis (VOC) is the primary reason for emergency medical care by SCD patients. Although neutrophils have been shown to play a role in the on-set of vaso-occlusion by interacting with sickle RBCs and platelets in cremaster venules of transgenic SCD mice, the cellular, molecular and biophysical mechanisms that promote vaso-occlusion in SCD patients is not completely understood. Materials and Methods: Freshly collected heparinized blood from steady-state SCD (SS) patients and race matched control subjects was perfused through polydimethylsiloxane (PDMS) based microfluidic flow channels (30 µm x 500 µm) with a glass bottom coated with either human microvascular endothelial cells or a cocktail of recombinant human P-selectin, ICAM-1 and IL-8 at a physiological shear stress (6 dyn cm-2). Fluorescent Abs against CD16 and CD49b were added to the blood for in-situ staining of neutrophils and platelets, respectively. Cellular interactions were recorded using quantitative microfluidic fluorescence microscopy (qMFM)1, which is a combination of quantitative dynamic footprinting1 and epifluorescence microscopy. Results and Discussion: Neutrophils in SS blood were observed to roll, arrest and then capture freely flowing platelets leading to the formation of vaso-occlusive aggregates. RBCs were observed getting trapped within the platelet-neutrophil aggregates. The number of platelet-neutrophil interactions, lifetime of these interactions and the extent of platelet-neutrophil aggregation were several folds higher in SS than control subject blood. Bacterial lipopolysaccharide (LPS; 500 ng/ml) pretreatment led to enhanced platelet-neutrophil aggregations in SS but not control blood. The enhanced platelet-neutrophil aggregations in SS blood (+/-LPS) was attenuated to the level observed in control blood by simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils with functional blocking Abs. Conclusion: Our data demonstrates that the vaso-occlusive pathophysiology in SCD involves sequential steps of neutrophil arrest, nucleation of platelets on arrested neutrophils, formation of platelet-neutrophil aggregates and trapping of RBCs in these aggregates. The inflammatory milieu of SS patient blood sets a lower threshold for bacterial endotoxin induced platelet-neutrophil aggregation than control blood. Vaso-occlusion can be ameliorated in SS blood by simultaneous inhibition of platelet P-selectin and neutrophil Mac-1. Understanding the molecular mechanism of vaso-occlusion will enable the development of therapies that can prevent VOC in SS patients. References: 1. Jimenez MA, Tutuncuoglu E, Barge S, Novelli EM, Sundd P. Quantitative microfluidic fluorescence microscopy to study vaso-occlusion in Sickle Cell Disease. Haematologica, 2015. 2 Sundd, P. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nat Methods 7, 821-824, doi:10.1038/nmeth.1508 (2010). Disclosures No relevant conflicts of interest to declare.

scholarly journals GTx011, a Potent Allosteric Modifier of Hemoglobin Oxygen Affinity, Prevents RBC Sickling in Whole Blood and Prolongs RBC Half-Life in Vivo in a Murine Model of Sickle Cell Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 217-217 ◽  
Author(s):  
Kobina Dufu ◽  
Donna Oksenberg ◽  
Chengjing Zhou ◽  
Athiwat Hutchaleelaha ◽  
David R. Archer
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Half Life ◽  
Ex Vivo ◽  
Cell Disease ◽  
Employment Equity ◽  
Hypoxic Conditions ◽  
Equity Ownership

Abstract Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene leading to production of hemoglobin S (HbS) that polymerizes under hypoxic conditions with subsequent formation of sickled red blood cells (RBCs). We have developed a novel small molecule, GTx011, which attains effective concentrations in blood upon oral dosing in multiple species. GTx011 increases the affinity of oxygen (O2) for HbS, delays in vitro HbS polymerization and prevents sickling of isolated RBCs under hypoxic conditions. We report here that GTx011 prevents in vitro sickling of RBCs in blood from sickle cell patients. Moreover, in a murine model of sickle cell disease (Townes SS mice), GTx011 prevents ex vivo sickling of RBCs and prolongs RBC half-life. We previously reported that GTx011 prevents sickling of isolated sickle cell RBCs (SSRBCs) subjected to a fixed hypoxic condition (pO2 of ~30 mm Hg) for 30 min. For a more physiologically relevant evaluation, we determined the anti-sickling activity of GTx011 in blood under variable hypoxic conditions over a shorter duration of time. Sickling of SSRBCs in blood was evaluated using a combination of hemoximetry and morphometric measurements. Whole blood from sickle cell patients was modified in vitro with GTx011 prior to hemoximetry. Conversely, blood from SS mice with GTx011 orally dosed acutely or chronically for 10-12 days was used for hemoximetry. SSRBCs were harvested during hemoximetry at various O2 tensions and immediately fixed in a deoxygenated solution of 2% glutaraldehyde/PBS prior to morphological quantitative analysis with CellVigene software or imaging flow cytometry (AMNIS ImageStreamX MkII). To evaluate the effect of GTx011 on RBC half-life in SS mice, N-hydroxysuccinimide biotin was injected into SS mice on day 5 of chronic dosing, producing a pulse-label. Flow cytometry was performed using fluorescently labeled streptavidin to determine the decay of biotinylation and RBC half-life. Reticulocyte counts were measured at different intervals during the dosing regimen by determining the percentage of blood cells that were Ter-119+, Thiazole-Orange+ and CD45- by flow cytometry. In a dose-dependent manner, GTx011 decreased the p50 value of human blood indicating an increase in Hb-O2 affinity. In parallel, GTx011 dose-dependently reduced the number of sickled SSRBCs under all hypoxic conditions (pO2 of <40 mm Hg) evaluated. Moreover, at an O2 tension mimicking typical hypoxic conditions in tissue capillaries (40 mm Hg), 300 µM of GTx011 was sufficient to prevent sickling of human SSRBCs in whole blood (20% Hct). Similarly, ex vivo sickling analysis indicated that, relative to blood from vehicle-treated SS mice, blood from GTx011-treated SS mice showed a pronounced reduction in the number of sickled RBCs under hypoxic conditions with a concurrent reduction in p50. For example, at a pO2 of 10 mm Hg, 19% of SSRBCs in blood from GTx011-treated mice sickled ex vivo compared with 56% in blood from vehicle-treated SS mice. In SS mice chronically dosed with GTx011, a prolongation of the RBC half-life from 2.4 days to 3.8 days was achieved together with a marked decrease in reticulocyte count. This increase in RBC half-life and accompanying reduction in reticulocyte count was observed in mice with GTx011 concentrations in blood that corresponded to >30% calculated Hb target occupancy. Taken together, these data suggest that GTx011 has the potential to be a beneficial therapeutic agent for the chronic treatment of SCD. Table SS mice RBC half life Reticulocytes Sickled RBCs Hemoximetry Chronic treatment, PO, BID, 10-12 days (Days) (%) (% at 10 mm Hg) p20 (mm Hg) p50 (mm Hg) Vehicle-treated 2.4 53 56 18 32 GBT440-treated (100mg/kg) 3.8 32 19 4.5 21 Disclosures Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Zhou:Global Blood Therapeutics: Research Funding. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Archer:Global Blood Therapeutics: Consultancy, Research Funding.

scholarly journals Voxelotor Improves Sickle Red Blood Cell Flow Under Hypoxia in a Microfluidic Venule

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Melissa Azul ◽  
David K. Wood
Keyword(s):  
Blood Flow ◽  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Microfluidic Device ◽  
Red Blood Cell ◽  
Cell Disease ◽  
Hypoxic Conditions ◽  
Flow Response ◽  
Gas Layer

Introduction Sickle cell disease affects a large population both nationally and globally. The disease is characterized by the presence of sickle hemoglobin, HbS, which polymerizes the red blood cell into a stiff, sickle shape upon deoxygenation. This polymerization causes several complications, most notably, vaso-occlusion. Voxelotor (Oxbryta, Global Blood Therapeutics) is a newly FDA approved therapeutic for the treatment of sickle cell disease that, when bound to HbS, maintains the oxy-Hb state and inhibits polymerization. Previous studies have demonstrated voxelotor's ability to improve the deformability of the sickle red blood cell (sRBC) via micropippeting and reduce viscosity under hypoxia through using a viscosmeter(Dufu et al, 2018), however its effect under dynamic flow conditions has yet to be explored. Microfluidic devices have served as useful tools to study sickle cell disease, allowing investigation under physiologic conditions of the rheological properties of the sRBC. In this experimental study we aim to examine voxelotor's effect on rheological properties of blood using a microfluidic platform that allows for direct observation of sickled blood flow in a physiologic relevant system. Materials and Methods Whole blood was drawn from 6 patients with sickle cell disease (HbSS or HbSC) as a part of routine blood work under an IRB approved protocol. The cohort included both pediatric and adult patients both on and off hydroxyurea. A stock solution of voxelotor in DMSO (dimethylsulfoxide) was mixed and stored in -20C until use. Red blood cells (RBCs) were isolated using centrifugation and fixed to 25% hematocrit with saline. Voxelotor was added to the blood samples for a final concentration of 500 uM. Voxelotor treated samples were then incubated at 37C for one hour. An untreated, non-incubated aliquot from each patient sample was also obtained to serve a control. From two patient samples, a DMSO vehicle control was also incubated at 37C for one hour to serve as an additional control. Using an electronic pressure regulator, blood from each treatment was then driven through a microfluidic device at a constant pressure and was exposed to hypoxic conditions while RBC velocity data was collected. The microfluidic device design and fabrication in this experiment is described in previously published studies(Wood et al, 2012; Valdez et al, 2019). Briefly, a 3-layer microfluidic device constructed of polydimethylsiloxane (PDMS) consists of a blood, hydration, and gas layer. Saline is perfused through the hydration layer to prevent blood evaporation throughout the experiment. Oxygen gas is pushed through the gas layer, exposing flowing blood to a specific oxygen tension achieved using a mixing setup supplied by air and nitrogen tanks. A fiber optic sensor records oxygen tension within the gas layer throughout the experiment. Deoxygenation-oxygenation cycles were conducted using oxygen saturations from 0 to 21% (0 to 160mmHg pO2). With each deoxygenation cycle after 0%, oxygen saturations were up titrated in a stepwise fashion until oxygen-independent flow was observed. RBC velocity was evaluated by tracking cell movement in the microchannel using high frame-rate imaging and computation video processing. Results and Conclusion A reduction in velocity occurs when sickle RBCs are exposed to deoxygenated conditions as seen in one sample example tracing in figure 1. However, the addition of voxelotor at 500 uM improved the blood flow response to deoxygenation, as RBCs treated with voxelotor had a reduction in velocity change compared to vehicle control and untreated samples when exposed to hypoxic conditions as low as 0 mmgHg oxygen (figure 2). Additionally, voxelotor treated samples began to experience oxygen-independent velocity at lower oxygen tensions compared to the controls. By inhibiting polymerization, voxelotor improves sensitivity of sickle RBC blood flow response in hypoxic conditions. While polymerization is one aspect of sickle cell disease, we would like to explore further effects of voxelotor on other aspects of the understood pathophysiology of the disease such as effects on adhesion in future experiments. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hypoxia Enhanced Red Cell Adhesion in Vitro May Identify Patients at Risk for Vasculopathy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3672-3672
Author(s):  
Charlotte Yuan ◽  
Erina Quinn ◽  
Sargam Kapoor ◽  
Myeongseop Kim ◽  
Erdem Kucukal ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Research Funding ◽  
Cell Disease ◽  
Hypoxic Conditions ◽  
Significant Difference ◽  
History Of ◽  
Male Subjects

Abstract Background: Priapism is a serious complication associated with Sickle Cell Disease (SCD) that may be a manifestation of underlying vasculopathy. The Centers for the Study of Complex Diseases of Childhood (CSCCD), comprising independent Comprehensive Sickle Cell Centers, demonstrated an association of priapism with hemolysis.1 Previously, we identified two groups of people with SCD based on red blood cell (RBC) adhesion under hypoxic conditions: those patients whose RBCs showed hypoxia-enhanced adhesion (HEA) and those whose did not (non-HEA).2 Patients with HEA had evidence for more hemolysis in vivo. Here, we aimed to examine (1) the association of HEA with hypoxia in vivo, and (2) RBC adhesion in normoxic and hypoxic conditions in male patients with or without a history of priapism. Methods: This retrospective study was conducted at the Adult Sickle Cell Disease Clinic at the University Hospitals Seidman Cancer Center, in Cleveland, OH between 2015 to 2018. Blood samples were obtained from 26 male subjects (29 samples, 25 HbSS and 1 HbSS HPFH). Adhesion experiments were performed as previously reported by passing surplus whole blood through LN-immobilized microchannels at physiological conditions under both normoxic and hypoxic conditions.2,3 Adherent RBCs were then quantified with microscope after a wash step. The median value was used for data analyses from multiple samples obtained from an individual. Chart review was conducted to examine results of hypoxia testing obtained in vivo as part of routine clinical care. Results: Male subjects with HbSS and a history of priapism had higher HEA in comparison to subjects without a history of priapism (3268 ± 5647 vs. 122 ± 1218, p=0.016). However, there was no significant difference between RBC adhesion of the two groups under normoxic conditions (529 ± 1528 vs. 402 ± 280). More male subjects with priapism had hypoxia in vivo (10 out of 14) than subjects without priapism (5 out of 12). Compared to male subjects with a history of priapism, those without a history of priapism had lower lactate dehydrogenase levels (474 ± 267 vs. 290 ± 215, p=0.008). Conclusions: Our data showed that subjects with a history of priapism had a higher HEA and tended to have more evidence for hypoxia in vivo than did subjects without a history of priapism. Further, male subjects with hypoxia in vivo had more HEA than did those without hypoxia in vivo (not shown). Hypoxia in vivo may cause increased RBC damage (reflected by HEA), hemolysis, nitric oxide depletion, and consequent vasculopathy, resulting in priapism. Hypoxia may be treatable, when identified in subjects with a history priapism in vivo or possibly with HEA in vitro. This could plausibly modify disease severity in some cases. References: Nolan VG, Wyszynski DF, Farrer LA, Steinberg MH. Blood. 20015 Nov;106(9):3264-7. doi: 10.1182/blood-2005-04-1594 Kim M, Alapan Y, Adhikari A, Little JA, Gurkan Microcirculation. 2017 Jul;24(5). doi: 10.1111/micc.12374. Alapan Y, Kim C, Adhikari A, Gray KE, Gurkan-Cavusoglu E, Little JA, Gurkan Transl Res. 2016 Jul;173:74-91.e8. doi: 10.1016/j.trsl.2016.03.008. Epub 2016 Mar 19. Disclosures Little: NHLBI: Research Funding; PCORI: Research Funding; Hemex: Patents & Royalties: Patent, no honoraria; Doris Duke Charitable Foundations: Research Funding.

scholarly journals Thromboinflammatory mechanisms in sickle cell disease - challenging the hemostatic balance

Haematologica ◽  
2020 ◽  
Vol 105 (10) ◽  
pp. 2380-2390 ◽  
Author(s):  
Nicola Conran ◽  
Erich V. De Paula
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ischemia Reperfusion ◽  
Organ Damage ◽  
Acute Chest Syndrome ◽  
Cellular Interactions ◽  
Innate Immune Responses ◽  
Cell Disease ◽  
Inflammatory Processes ◽  
Abnormal Hemoglobin

Sickle cell disease (SCD) is an inherited hemoglobinopathy that is caused by the presence of abnormal hemoglobin S (HbS) in red blood cells, leading to alterations in red cell properties and shape, as the result of HbS dexoygenation and subsequent polymerization. SCD pathophysiology is characterized by chronic inflammatory processes, triggered by hemolytic and vaso-occlusive events, which lead to the varied complications, organ damage and elevated mortality seen in individuals with the disease. In association with activation of the endothelium and leukocytes, hemostatic alterations and thrombotic events are well-documented in SCD. Here we discuss the role for inflammatory pathways in modulating coagulation and inducing platelet activation in SCD, due to tissue factor activation, adhesion molecule expression, inflammatory mediator production and the induction of innate immune responses, amongst other mechanisms. Thromboinflammatory pathways may play a significant role in some of the major complications of SCD, such as stroke, venous thromboembolism and possibly acute chest syndrome, besides exacerbating the chronic inflammation and cellular interactions that trigger vaso-occlusion, ischemia-reperfusion processes, and eventually organ damage.

scholarly journals Management of Hodgkin Lymphoma in a Sickle Cell Patient: A Case Report

2019 ◽  
Vol 12 (1) ◽  
pp. 224-227
Author(s):  
Farah Ashraf ◽  
Pragnan Kancharla ◽  
Mendel Goldfinger
Keyword(s):  
Quality Of Life ◽  
Case Report ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Blood Cells ◽  
Patient Population ◽  
White Blood Cells ◽  
Modern Medicine ◽  
Cell Disease

Sickle cell disease (SCD) is an inherited disorder of hemoglobin mutation in red blood cells, with a patient population that is increasing in age in recent decades due to advances in modern medicine. Hodgkin’s lymphoma (HL) is a cancer of white blood cells, and while concomitance of SCD and Hodgkin’s has been reported, a discussion of treatment for HL in SCD is lacking from the literature. We present a case of effectively treated HL in SCD and put forth that the regimen used is a practical choice, and as it was completed fully as outpatient, it improved the patient’s quality of life compared to an inpatient regimen.

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