scholarly journals Whole blood transcriptomic analysis reveals PLSCR4 as a potential marker for vaso-occlusive crises in sickle cell disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hawra Abdulwahab ◽  
Muna Aljishi ◽  
Ameera Sultan ◽  
Ghada Al-Kafaji ◽  
Kannan Sridharan ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Differentially Expressed ◽  
Enzyme Linked Immunosorbent Assay ◽  
Healthy Controls ◽  
Cell Disease ◽  
Blood Disorder ◽  
Potential Biomarker

AbstractSickle cell disease, a common genetic blood disorder, results from a point mutation in the β-globin gene affecting the configuration of hemoglobin, predisposing to painful vaso-occlusive crisis (VOC) and multi-organ dysfunctions. There is a huge variation in the phenotypic expressions of SCD and VOC owing to genetic and environmental factors. This study aimed to characterize the whole blood gene expression profile using Microarray technology in Bahraini patients with SCD determining the differentially expressed genes in steady-state (n = 10) and during VOC (n = 10) in comparison to healthy controls (n = 8). Additionally, the study intended to identify potential genetic marker associated with hemolysis. The analysis identified 2073 and 3363 genes that were dysregulated during steady-state and VOC, respectively, compared to healthy controls. Moreover, 1078 genes were differentially expressed during VOC compared to steady state. The PLSCR4 gene was almost 6-fold up-regulated in microarray, 4-fold in polymerase chain reaction, and a mean protein concentration of 0.856 ng/ml was observed in enzyme-linked immunosorbent assay during VOC compared to steady-state (0.238 ng/ml) (p < 0.01). Amongst these genes, PLSCR4 is involved in erythrocyte membrane deformity thus, predisposing to hemolysis, adhesion, and thrombosis. In conclusion, PLSCR4 may serve as a potential biomarker for VOC and future large-scale validation are recommended.

Analysis Of Hemostatic Characteristics using Thromboelastometry in Adults With Sickle Cell Disease and Controls

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4766-4766
Author(s):  
Maissaa Janbain ◽  
Cindy A. Leissinger ◽  
Rebecca Kruse-Jarres
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Sickle Cell Trait ◽  
Acute Illness ◽  
Cellular Component ◽  
Healthy Controls ◽  
Cell Disease ◽  
The Impact

Background Vaso-occlusive phenomena and hemolysis are the clinical hallmarks of sickle cell disease (SCD). In addition, pain crisis was identified as the initial clinical manifestation in 61.9% of sickle cell patients who died shortly after hospital admission from thromboembolism and micro vascular thrombi.  Knowing that the vaso-occlusive events may be related to activation of the hemostatic system, and that thromboelastometry (TEM) assesses the functionality of this system from a global standpoint, it will be challenging to characterize the findings in patients with SCD as a way to differentiate their clinical phenotype upon presentation, and to predict the impact of these manifestations on their prognosis, mortality and morbidity. Objective   To characterize the findings of TEM in patients with SCD during periods of steady state and acute illness, to compare these results with those of healthy controls and sickle cell trait (SCT), to compare the findings in whole blood (WB) to plasma (PL) for each category, in a way to analyze the findings in plasma and their applicability in clinical practice as well as to delineate the contribution of the cellular component in whole blood samples. Design  In a cross-sectional study, we obtained TEM and other hemostatic data on 24 adult patients with SCD (16 in steady state and 8 in acute illness); and 13 race and age matched healthy controls (6 with sickle cell trait (SCT) and 7 with no trait). We specifically studied coagulation time (CT) as a function of coagulation factors; clot firmness time (CFT) and alpha angle(α) assessing platelet and fibrinogen function; and maximum clot firmness (MCF) evaluating the mechanical clot quality (plt, fibrinogen and factorXIII) and finally thrombodynamic potential index (TPI) as a function of patient’s global coagulation. Results   Overall, patients with SCD had higher TPI in WB (p=0.23;=0.25) and lower TPI in PL (p<0.0001;=0.47) when compared to controls and SCT respectively. Also, patients with SCD had lower CT (p=0.02), lower CFT (p<0.0001) higher MCF; α and TPI (p<0.0001; =0.051; <0.0001) in whole blood compared to plasma. Sickle cell trait patients had lower CT, CFT, alpha with higher MCF in WB compared to PL. While healthy controls had higher CT; MCF and TPI (p=0.01; <0.0001; <0.0001) and lower CFT, α (p=0.16; <0.0001) in WB compared to PL. Conclusion  Whole blood of SCD patients seems to be hypercoagulable in comparison to WB of controls and SCT. While the plasma of SCD patients was significantly hypocoagulable when compared to PL of healthy controls. Overall, TEG profiles of WB were different than PL. This was more obvious in SCD patients reinforcing the contribution of the cellular component to the pathophysiology of this disease and the possible compensatory hypocoagulable status of the plasma in these patients. Further study of larger and more homogeneous patient groups, is required to adequately assess the clinical utility of TEM in patients with sickle cell disease. Disclosures: Kruse-Jarres: Baxter Healthcare: Consultancy; Bayer HealthCare: Consultancy; Biogen IDEC: Consultancy; Grifols: Consultancy; Kedrion: Consultancy; Novo Nordisk: Consultancy.

scholarly journals Cathepsin B, a Negative Regulator of Autophagy, Identified As a Novel Therapeutic Drug Target in Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Jagadeesh Ramasamy ◽  
Vinzon Ibanez ◽  
Kareem Al-Qadi ◽  
Helen Zhang ◽  
Jennifer Afranie-Sakyi ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Western Blot ◽  
Sickle Cell ◽  
Cathepsin B ◽  
Negative Regulator ◽  
Differentially Expressed ◽  
Therapeutic Drug ◽  
Cell Disease ◽  
Blood Disorder ◽  
Autophagy Pathway

Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide. The disease is caused by a mutation of the beta-globin gene that results in polymerization of the sickle hemoglobin (HbS) when deoxygenated. Reactive oxygen species (ROS) induced hemolysis is a major critical event in SCD. We have previously shown that abnormal retention of mitochondria in the erythrocytes of both SCD patients and SCD mice is associated with elevated ROS levels and hemolysis (Exp. Hem.2017;50:46-52). The mechanism responsible for mitochondrial retention in SCD is unknown. Autophagy is one of the processes responsible for the elimination of mitochondria during erythroid differentiation. Autophagy is a conserved physiological process that promotes cellular homeostasis through the recycling of proteins, protein aggregates, and removal of damaged organelles. Hypothesis: In this study, we have investigated the hypothesis that autophagy pathway dysregulation is responsible for abnormal mitochondrial retention in SCD erythrocytes. We propose that pharmacological methods that address the abnormal activity of these genes may be therapeutic for SCD. Methods: The expression of autophagy pathway genes in reticulocytes of SCD patients and experimental SCD mice was compared with their respective controls using the RT2 Autophagy Profiler PCR Array. The expression of autophagy proteins was measured by Western blot. Cathepsin B (CTSB) activity was measured by a fluorometric assay using the substrate RR-AFC (amino-4-trifluoromethyl coumarin). Results: Six autophagy pathway genes were differentially expressed in SCD patients compared to control subjects. Expression of the autophagy pathway genes was also analyzed in SCD mice, normal AA mice, and phlebotomized anemic AA mice as a control for increased reticulocytosis. Eighteen genes were differentially expressed in SCD mice compared with control AA mice (p&lt;0.05). Four genes were differentially expressed in SCD mice compared with control phlebotomized anemic AA mice (p&lt;0.05). CTSB, a known negative regulator of autophagy (J Exp Med. 2016;213(10):2081-97), was over-expressed in the reticulocytes of both SCD patients and SCD mice. Expression of CTSB was 4.5 fold (p&lt;0.007) higher in SCD mice compared to normal control AA mice and 8.6 fold (p&lt;0.001) higher compared to phlebotomized AA anemic mice. We therefore focused our further analysis on CTSB. Western blot data confirmed that CTSB protein was overexpressed in red blood cells of SCD patients and experimental SCD mice (p&lt;0.01) compared to respective controls. The level of CTSB enzyme activity was 3.3 fold higher in SCD mice compared to the control mice(p&lt;0.01). To evaluate the functional significance of these differences in CTSB expression, CTSB inhibitor E64d was investigated in SCD mice. SCD mice treated with E64d (40mg/kg b.wt.) for two weeks showed a reduction in CTSB activity (SCD vehicle: 11.8±1.1 RFU/µg, n=3 vs SCD E64d treatment: 5 ± 0.1 RFU/µg, n=3, p&lt;0.008) and in the proportion of reticulocytes that retained mitochondria (SCD vehicle: 45.2%±3%, n=3 vs. SCD E64d treatment: 32 % ± 1.9%, n=3, p&lt;0.003). Reticulocytosis was also significantly reduced in SCD mice treated with the CTSB inhibitor (SCD vehicle: 47.2%±2.8%, n=3 vs. SCD E64d treatment: 35.9 % ± 3.9%, n=3, p&lt;0.01). Conclusion: Our data shows that the multiple autophagy pathway gene expression is dysregulated in SCD. Among these genes, CTSB, a key negative regulator of autophagy, is overexpressed in the reticulocytes of both SCD patients and SCD mice. Pharmacological inhibition of CTSB activity partially reverses mitochondrial retention and decreases reticulocytosis in SCD mice, confirming that overexpression of CTSB contributes to SCD pathogenesis and is a potential therapeutic target. Disclosures No relevant conflicts of interest to declare.

scholarly journals Circulating Cell-Free DNA in Sickle Cell Disease: Is It a Potentially Useful Biomarker?

2014 ◽  
Vol 138 (5) ◽  
pp. 678-683 ◽  
Author(s):  
Salah Al-Humood ◽  
Rajaa Zueriq ◽  
Lama Al-Faris ◽  
Rajaa Marouf ◽  
Fahd Al-Mulla
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Plasma Cell ◽  
Sickle Cell Trait ◽  
Healthy Controls ◽  
Cell Disease ◽  
Cell Free Dna ◽  
Free Dna ◽  
Polymerase Chain

Context.—Vascular occlusion in sickle cell disease causes increased levels of plasma cell-free DNA as a result of cell death and tissue damage. Objectives.—This study investigates plasma cell-free DNA concentrations in sickle cell disease patients, and aims at exploring the significance of plasma cell-free DNA as a potential biomarker in predicting its complications. Design.—Plasma cell-free DNA levels were measured using real-time quantitative polymerase chain reaction to quantitatively measure β-globin gene in blood samples from 57 sickle cell disease patients with acute vaso-occlusive crisis, 42 patients in steady state, 16 individuals with sickle cell trait, and 40 healthy controls. Results.—Plasma cell-free DNA level was significantly elevated in samples from patients with acute vaso-occlusive crisis when compared with those in steady state (P = .002), and was significantly higher both in crisis and in steady state when compared with individuals with sickle cell trait and healthy controls (P &lt; .001). There was no difference in cell-free DNA levels between individuals with sickle cell trait and healthy controls. There was no association between plasma cell-free DNA levels and various clinical complications of sickle cell disease and comorbidity. Conclusions.—Plasma cell-free DNA, as quantified by polymerase chain reaction amplification of the β-globin and human telomerase reverse transcriptase genes, is increased in sickle cell disease patients in vaso-occlusive crisis and in steady state compared with individuals with sickle cell trait and healthy controls, and may be used as a tool to diagnose and monitor the sickle cell crisis and differentiate post–packed red cell transfusion sickle cell disease patients from individuals with sickle cell trait.

scholarly journals Type I Interferon Gene Signature in Peripheral Blood Mononuclear Cells of Sickle Cell Disease Patients and a Connection to RBC Alloimmunization

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-27
Author(s):  
Emaan Madany ◽  
June Young Lee ◽  
Jeanne E. Hendrickson ◽  
David R Gibb
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Gene Signature ◽  
Small Sample ◽  
Type I ◽  
Healthy Controls ◽  
Cell Disease ◽  
Interferon Gene

Background RBC alloimmunization is a clinically significant issue in transfusion medicine; patients with sickle cell disease have an increased risk of alloantibody production (30-50% of SS patients) compared to that of other hospitalized patients (3-10%). However, mechanisms underlying the increased frequency of alloimmunization in sickle cell patients are poorly understood. In previous studies, inflammation in the recipient has been shown to promote alloimmunization. Transfusion models, type 1 interferons (IFNα/β) and Interferon Stimulated Genes (ISGs) have been shown to promote alloimmunization in mice. Other studies have shown that patients with inflammatory autoimmune diseases express an IFNα/β signature which may contribute to the increased frequency of alloimmunization in these populations. One recent study reported significantly elevated ISGs in neutrophils as well as evidence that IFNα is upregulated in SS patients compared to controls. Given the chronic inflammatory state in SS patients, we sought to determine the role of PBMCs and whether they also expressed an IFN gene signature that contributes to the increased frequency of alloimmunization. Methods The expression of the ISG, myxovirus resistance protein 1 (MxA), was measured in the blood of SS patients with more patients with SS disease (SS, n=13) and race matched healthy controls (ββ, n=3) by whole blood immunoassay (ELISA). qPCR was performed on 5 previously established ISGs to determine an IFN score, a measure of overall gene expression, from whole blood and IFNβ stimulated PBMCs of SS patients (SS, n=15) and healthy race matched controls (ββ, n=5). A LEGENDplex™ Human Anti-Virus Response Panel assay was used to determine the expression of various type 1 IFNs, cytokines and ISGs in patients with SS disease (SS, n=15) and healthy race matched controls (ββ, n=5). Results SS patients had significantly elevated levels of MxA (mean ± standard error of the mean, SS MxA = 12.27 ng/mL ± 15.68) compared to control patients without SS (ββ MxA = 1.52 ng/mL± 0.26, p&lt; 0.05) (Figure 1 A) . The Legendplex showed a significant increase in IL-6, IL-10 and the ISG, IP-10. (SS IP-10= 147.81 pg/mL ± 49.24) (ββ IP-10 = 68.85 pg/mL ±10.70, p&lt;0.01) (Figure 1 B) Analyzing the 5 ISGs, we saw a trend towards a higher IFN score in patients with SS disease than healthy controls in whole blood; this difference was significant in PBMCs stimulated with IFNB (IFN Score SS = 20.76 ± 17.18 ,ββ = 0.00 ± 3.71 , p&lt;0.01) (Figure 1 C). Discussion SCD is a complex disease with many environmental and genetic factors that play roles in the severity of the disease. Any number of these factors may influence the high rates of alloimmunization found in sickle cell patients. We found increased cytokines, ISGs and IFN scores in SS patients compared to healthy controls. These findings suggest the presence of an IFNα/β gene signature in patients with sickle cell disease. Due to the relatively small sample size, we are unable to determine a correlation between alloantibodies and MxA levels or high IFN scores with this cohort. Further studies will allow us to determine if the increased interferon gene signature plays a role in the increased alloimmunization burden that these patients experience. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cell-Free Mitochondrial DNA Is Elevated in Sickle Cell Disease Patients, and Serve As a Potential Proinflammatory DAMP

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1068-1068
Author(s):  
Laxminath Tumburu ◽  
Shohini Ghosh-Choudhary ◽  
Emilia Alina Barbu ◽  
Simon Yang ◽  
Lauren D Harrison Ware ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Acute Pain ◽  
Organ Damage ◽  
Healthy Controls ◽  
Cell Disease ◽  
Cross Sectional ◽  
Healthy Donors

Abstract Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by hemolysis and intermittent acute pain with multi-organ damage. Previously, we showed that acute pain in SCD was associated with >10-fold increases in cell-free DNA (cfDNA) when compared to steady state, that were significantly reduced during hydroxyurea therapy. Apoptosis, necrotic cell death and lysis of intact cells in the blood stream have been proposed as sources of plasma cfDNA. Here, we explored if the cfDNA increases could have a role in inflammation, a constant pathological feature of SCD. cfDNA was extracted using QIAamp MinElute ccfDNA Kit (Qiagen), from the platelet-poor plasma processed within 30 minutes from the blood drawn in EDTA tubes, and analyzed using whole genome sequencing (WGS) and targeted quantitative PCR (qPCR). SCD patients are defined as in acute pain if there is no evident cause other than SCD, for which the patient needs hospitalization, either as in- or outpatient, and is treated with parenteral narcotics. Steady state was defined as the period from at any time 8 weeks prior to or after a crisis. A cross-sectional study of 8 healthy controls and 34 SCD patients (18 steady-state; 16 crisis) mapped WGS reads showed significantly higher proportion of cell-free mitochondrial DNA (cf-mtDNA) compared to nuclear cfDNA (cf-nDNA) in SCD patients compared with healthy controls (Fig 1A: steady-state: 14 fold; crisis: 11 fold; p = 0.0001). We used targeted qPCR to quantify both cf-nDNA and cf-mtDNA in another cross-sectional cohort of 13 healthy controls and 92 patients (72 steady-state, 20 crisis) as well as 18 paired HbSS patients (steady-state and crisis) samples with 10 healthy controls. The nuclear reference genes used were GAPDH and TERT and mitochondrial genes were MT-ND1 and MT-ND6. While cf-nDNA (TERT) was significantly increased (> 3.5 fold, p = 0.0251; Fig 1B) in SCD patients compared with healthy controls only during crises, significantly higher levels of cf-mtDNA over cf-nDNA were observed in SCD patients compared with healthy volunteers in both steady-state and crises (Fig 1C: MT-ND1/GAPDH: steady-state >19 fold, crisis > 8 fold; MT-ND1/TERT: steady-state > 8 fold, crisis > 7 fold; MT-ND6/GAPDH: steady-state > 7 fold, crisis > 3 fold; MT-ND6/TERT: steady-state > 4 fold; crisis > 4 fold; p < 0.05). In the paired samples, cf-nDNA (GAPDH andTERT) was significantly increased (> 3 fold; Fig 1D-E) in crisis compared to steady-state (p < 0.05). The differential increase in cf-mtDNA (cf-mtDNA:cf-nDNA ratio) levels in these patients during crises, were significantly higher compared with healthy controls (Fig 1F: MT-ND1/GAPDH: steady-state >9 fold, crisis > 8 fold; MT-ND1/TERT: steady-state > 8 fold, crisis > 9 fold; MT-ND6/GAPDH: steady-state > 8 fold, crisis > 8 fold; MT-ND6/TERT: steady-state > 8 fold; crisis > 7 fold; p < 0.005). Using confocal microscopy and mitochondrial-specific dyes (MitoTracker Green and TMRM), we show that substantial numbers of red blood cells from SCD patients retain their mitochondria in the circulation. We next explored if the elevated cf-mtDNA in SCD could contribute to its pathophysiology, via activating neutrophils to form neutrophil extracellular traps (NETs), a recognized immunological response in inflammation. Initially, we confirmed that mtDNA can induce NETosis by treating neutrophils from healthy donors with mtDNA isolated from human platelets. mtDNA consistently induced a robust NETs response (N=8) while genomic nuclear DNA did not cause any NETosis. SCD plasma containing high levels of cf-mtDNA also caused a strong NETosis response while plasma from healthy donors did not (N=11). Cytosolic adaptor STING has a central role in sensing of cytosolic double stranded DNA. We sought to determine if the downstream STING-TBK1-IRF3 pathway is associated with the mtDNA-mediated formation of NETs. We inhibited the catalytic activity of the STING downstream effector TBK1 with BX795 prior to treating neutrophils with cf-mtDNA-containing plasma (N=5). The TBK1 inhibition consistently reduced the NETs response by at least 70% confirming that cytosolic DNA sensors are involved in promoting mtDNA-mediated formation of NETs. Our findings suggest that cf-mtDNA induces NETosis contributing to the pathological sterile inflammation in SCD patients. Continual release of these mitochondrial DAMPs in hemolysis may serve as key link between inflammation and organ damage in SCD. Disclosures No relevant conflicts of interest to declare.

Placenta growth factor activates monocytes and correlates with sickle cell disease severity

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1506-1514 ◽  
Author(s):  
Natalya Perelman ◽  
Suresh K. Selvaraj ◽  
Sandeep Batra ◽  
Lori R. Luck ◽  
Anat Erdreich-Epstein ◽  
...  
Keyword(s):  
Steady State ◽  
Growth Factor ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Mononuclear Cells ◽  
Mrna Levels ◽  
Healthy Controls ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Placenta Growth Factor

Abstract Sickle cell disease (SCD) results in chronic hypoxia and secondarily increased erythropoietin concentrations. Leukocytosis and activated monocytes are also observed in SCD in absence of infection or vaso-occlusion (steady state), the reasons for which are unknown. We found that erythroid cells produced placenta growth factor (PlGF), an angiogenic growth factor belonging to the vascular endothelial growth factor (VEGF) family, and its expression was induced in bone marrow CD34+ progenitor cells in the presence of erythropoietin. Furthermore, the steady state circulating PlGF levels in subjects with severe SCD (at least 3 vaso-occlusive crises [VOCs] per year) were 18.5 ± 1.2 pg/mL (n = 9) compared with 15.5 ± 1.2 pg/mL (n = 13) in those with mild SCD (fewer than 3 VOCs per year) and 11.3 ± 0.7 pg/mL (n = 9) in healthy controls (P &lt; .05), suggesting a correlation between PlGF levels and SCD severity. In addition, PlGF significantly increased mRNA levels of the proinflammatory cytochemokines interleukin-1β, interleukin-8, monocyte chemoattractant protein-1, and VEGF in peripheral blood mononuclear cells (MNCs) of healthy subjects (n = 4; P &lt; .05). Expression of these same cytochemokines was significantly increased in MNCs from subjects with SCD at steady state (n = 14), compared with healthy controls. Of the leukocyte subfractions, PlGF stimulated monocyte chemotaxis (P &lt; .05, n = 3). Taken together, these data show for the first time that erythroid cells intrinsically release a factor that can directly activate monocytes to increase inflammation. The baseline inflammation seen in SCD has always been attributed to sequelae secondary to the sickling phenomenon. We show that PlGF contributes to the inflammation observed in SCD and increases the incidence of vaso-occlusive events.

scholarly journals A Standardized Clinical Flow Adhesion Assay of Whole Blood Adhesion to VCAM-1 Predicts Severe SCD Phenotypes in a 2-Yr Prospective Observational Follow-up of the Original Elipsis Cohort

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3562-3562
Author(s):  
Patrick C. Hines ◽  
Ahmar Urooj Zaidi ◽  
Xiufeng Gao ◽  
Ke Liu ◽  
Muhammad O. Shareef ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Hospital Admissions ◽  
Phenotypic Variability ◽  
Acute Chest Syndrome ◽  
Adhesion Assay ◽  
Cell Disease ◽  
Equity Ownership

Sickle cell disease (SCD) is a complex, multi-organ system condition characterized by frequent and unpredictable vaso-occlusive episodes (VOEs) and significant phenotypic variability. Red blood cell (RBC) adhesion contributes to vaso-occlusive pathology, thus we have developed and validated a standardized clinical whole blood flow adhesion assay to VCAM-1 (FA-WB-VCAM) to serve as a clinical biomarker of RBC health in sickle cell disease and other conditions1-4. Blood for the FA-WB-VCAM assay is drawn in sodium citrate tubes and can be stored at 4oC for up to 48hrs. Whole blood samples are perfused through VCAM-1-coated channels and adherent blood cells are quantified manually to generate a clinical adhesion index (cells/mm2). We previously reported longitudinal steady-state FA-WB-VCAM adhesion indices established from every 3 weeks blood sampling over 6 months (mean=368.3 ±198.4 cells/mm2, range=83.50 - 917.0), and the correlation of steady state adhesion indices to a lifetime historical SCD severity index (SCDI) (r=0.5851, p=0.0003) 5,6. The lifetime historical SCDSI was calculated by quantifying the total number of objectively, documentable, vaso-occlusive end-organ events (VEEs), including acute chest syndrome/pneumonia, stroke, priapism, splenic sequestration, hepatic sequestration, and cholelithiasis, indexed over the total years of life (# VEEs/age; range=0.0 to 0.38 in ELIPSIS). The objective of this study was to determine if the FA-WB-VCAM could predict clinical SCD severity prospectively using various metrics of disease severity, including the SCDI over a 2-year period post ELIPSIS. First, we classified study subjects as "severe" or "mild/moderate" adhesion phenotypes defined by mean steady state adhesion indices acquired from every 3 week sampling over 6 months of the ELIPSIS study (severe adhesion phenotype: &gt; 75th percentile, 455.9 cells/mm2; mild/moderate adhesion phenotype: &lt; 75thpercentile). VEEs were verified by retrospective review of medical records, and a SCDSI over the 2-year period following the ELIPSIS study was established. Our data show that individuals with severe adhesive SCD phenotypes experienced significantly more VEEs compared to individuals with low/moderate severe adhesive SCD phenotypes (mean=55.67 ±69.78 compared to mean=17.04 ±20.58 respectively; p=0.01). SCD patients with severe adhesive phenotypes also had more hospital admissions (mean=5.67 ±2.29 compared to mean=2.88 ±3.41, p=0.001), ER visits (mean=36.00 ±63.42 compared to mean=9.20 ±15.65, p=0.02), transfusions (mean=4.33 ±3.74 compared to mean=1.60 ±2.55, p=0.01), acute chest syndrome/pneumonia (mean=1.56 ±0.73 compared to mean=0.20 ±0.65, p&lt;0.001), and priapism (mean=2.56 ±5.57 compared to mean=0.00 ±0.00, p=0.003) when compared to low/moderate adhesive phenotypes. These data suggest that the FA-WB-VCAM assay may serve as a predictive biomarker for impending VEEs, and a monitoring biomarker to assess response to SCD-modifying therapies. Additional studies in a larger prospective cohort are required to definitively establish the clinical utility of the FA-WB-VCAM assay. Disclosures Hines: Functional Fluidics: Equity Ownership. Gao:Functional Fluidics: Equity Ownership. Liu:Functional Fluidics: Employment. White:Functional Fluidics: Equity Ownership.

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

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

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

Constitutive Activation of Hemostasis in Children with Sickle Cell Disease.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1216-1216
Author(s):  
MacGregor Steele ◽  
Suzan Williams ◽  
Fred Pluthero ◽  
K.W. Annie Bang ◽  
Ran Goldman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Surface Expression ◽  
Hypercoagulable State ◽  
Cell Disease ◽  
Normal Children

Abstract Recent studies of sickle cell disease (SCD) suggest that hemostatic activation is a key aspect of pathophysiology, leading to clinical consequences such as vaso-occlusive crises and stroke. In many of these studies a SCD-associated hypercoagulable state has been inferred from markers suggestive of increased hemostatic activity, such as elevated levels of plasma thrombin-antithrombin complexes and tissue factor-containing microparticles. As part of a study using a broad-spectrum approach to explore the relationships among various aspects of normal and abnormal hemostasis in SCD we used a whole-blood coagulation assay (thromboelastography, TEG) to directly assess global hemostatic activation in children with SCD defined by genotype (SS and SC), together with a group of unaffected children. We also assessed platelet activation and procoagulant surface expression in platelets and red blood cells (RBCs) using flow cytometry. Eligible SCD subjects included:patients with painful crisis assessed at two time points: hospital admission (crisis group) and clinic follow-up appointment (follow-up group);patients not in crisis attending a regular clinic appointment (steady-state group). Patients were ineligible if they had received a recent blood transfusion, hydroxyurea, anticoagulants or aspirin. The results of TEG assays with citrated whole blood showed that compared to SC patients (n = 16) and normal children (n = 16), SS patients (n = 45) had significantly (p&lt;0.001) earlier clotting onset (mean R times were 4.5, 6.5 and 7.6 minutes for SS, SC and normals respectively) and significantly (p&lt;0.001) higher rates of clotting (mean maximum clotting rates were 16.8, 12.6 and 10.9 mm/min for SS, SC and normals respectively). TEG clotting onset and maximum clotting rates were not significantly different among steady-state, crisis and follow-up groups of children with SCD (both SS and SC genotypes), nor between sexes within each study group. Whole blood flow cytometry revealed that platelet GPIIb/IIIa activation (PAC-1 binding) was significantly elevated (p&lt;0.05) in SCD patients relative to normal children. In addition, markers of RBC procoagulant surface expression (RBC annexin A5 binding) and RBC-platelet aggregates were elevated in SCD patients compared to normal children. These results indicate that children with the SS genotype have an activated hemostatic system relative to normal and SC genotype children, and that this hypercoagulable state is maintained during sickle cell crisis as well as during steady state. It remains to be determined whether pharmacological interventions and/or RBC transfusions which improve clinical outcomes in SCD patients modify their constitutively hypercoagulable state.

scholarly journals The Chft+ Sensor: A Novel Method to Measure Perfusion Abnormalities in Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-9
Author(s):  
Anna Shvygina ◽  
Ali R. Roghanizad ◽  
Thomas Diller ◽  
Emily Moses ◽  
Vydia Permashwar ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Healthy Volunteers ◽  
Sickle Cell ◽  
The Body ◽  
Minimal Amount ◽  
Healthy Controls ◽  
Peripheral Perfusion ◽  
Cell Disease ◽  
Potential Biomarker ◽  
Perfusion Deficits

Introduction: Peripheral perfusion limitation is a potential biomarker of the severity and progression of sickle cell disease (SCD). Currently, there exists no direct means of quantifying perfusion deficits in SCD patients. Transcranial doppler ultrasonography offers a surrogate measure of perfusion and is highly operator dependent. Routine clinical screening of perfusion abnormalities with alternative modalities (e.g., positron emission tomography and magnetic resonance imaging) is challenging given high cost, requirement for sedation in young children, and need for trained personnel. The Combined Heat-Flux Temperature Sensor (CHFT+) is a novel noninvasive bio-heat perfusion sensor that measures peripheral perfusion in real time at depths of up to 1 cm by applying a minimal amount of heat to the skin surface. Because perfusion limitation is a hallmark of SCD, an improved understanding of its pathophysiology could potentially empower both patients and their physicians. The aim of this study was to evaluate the ability of the CHFT+ to detect differences in peripheral perfusion between healthy volunteers and patients with SCD. Materials and Methods: The present study is an ongoing, non-randomized pilot study that aims to test the CHFT+ sensor's ability to detect impaired perfusion is pediatric SCD patients. We are actively enrolling pediatric patients (age 3-17) with SCD and healthy volunteers from 2 pediatric clinics to test the sensor. Peripheral perfusion measurements were obtained at 5 locations on the body (e.g., forehead, forearms, palms). All measurements were normalized against forehead perfusion values to account for individual variations. An independent-samples t-test was conducted to compare average rates of perfusion in patients with SCD and in healthy volunteers. The goal enrollment for this study is 52 patients (26 with SCD, 26 healthy controls) to detect a two-fold difference in perfusion with a power of 0.80. Interim Results and Discussion: To date, 31 participants have been enrolled, 10 (32%) of whom were patients with SCD. Overall, average perfusion rates were lower in the SCD group when compared to the healthy group, although these differences were not statistically significant. Representative measurements of perfusion in the right and left forearms, normalized against forehead rates, are shown in Figure 1. Normalized left forearm perfusion in healthy controls vs. SCD patients: avg = 1.77, SD = 0.95 vs. avg = 1.28, SD = 0.52, respectively [t(27) = 1.80, p = 0.08]; see Fig. 1a. Normalized right forearm perfusion in healthy controls vs. SCD patients: avg = 1.80, SD = 1.01 vs. avg = 1.31, SD = 0.60, respectively [t(25) = 1.55, p = 0.13]); see Fig. 1b. Conclusions:At this time, our data suggest that the novel CHFT+ sensor is capable of detecting a modest difference in peripheral perfusion in children with SCD versus healthy controls, but this difference is not statistically significant due to limited enrollment. Additional enrollment will further define the utility of the CHFT+ sensor in this clinical population. Future studies should evaluate the sensor's capability to detect perfusion deficits in the adult SCD population, as well as measure perfusion variations that occur during SCD vaso-occlusive crises. Disclosures Diller: FluxTeq:Other: Partner.

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