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

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.

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

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2012-0725-oa ◽

2014 ◽

Vol 138 (5) ◽

pp. 678-683 ◽

Cited By ~ 7

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 < .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.

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

Scientific Reports ◽

10.1038/s41598-021-01702-8 ◽

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.

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Hypoxia Impact on Red Blood Cell-Mediated Microvascular Occlusion and Adhesion in Sickle Cell Disease and Sickle Cell Trait

Blood ◽

10.1182/blood-2021-149121 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 966-966

Author(s):

Yuncheng Man ◽

Zoe Sekyonda ◽

Karamoja Monchamp ◽

Ran An ◽

Erdem Kucukal ◽

...

Keyword(s):

Sickle Cell Disease ◽

Blood Cell ◽

Sickle Cell ◽

Red Blood Cell ◽

Whole Blood ◽

Sickle Cell Trait ◽

Single Gene ◽

Cell Disease ◽

The Impact ◽

Rbc Deformability

Abstract Introduction: Sickle cell disease (SCD) is a recessively inherited anemia caused by a single gene mutation leading to sickle hemoglobin production. Sickle cell trait (SCT) is the carrier state. Abnormal hemoglobin polymerization and resultant red blood cell (RBC) sickling, decreased deformability and increased adhesion, are well-known features of homozygous SCD. However, the overall pathophysiological impact of SCT on the RBC remains incompletely characterized. Here we use microfluidic techniques designed by us, the OcclusionChip and SCD Biochip (previously published), and commercially available ektacytometry to investigate hypoxia impact on RBC biophysical properties in SCT. Methods: Venous blood samples were collected in EDTA from subjects with homozygous HbSS, SCT (HbAS), and non-anemic controls (HbAA) under an IRB-approved protocol. OcclusionChip devices were fabricated using standard soft lithography protocols [1]. RBCs were isolated from whole blood, re-suspended in PBS at 20% hematocrit, and passed through the OcclusionChip device with a constant inlet pressure. Following a wash step, the OcclusionChip microchannel was imaged, and Occlusion Index (OI), a standardized generalizable parameter we developed, representing the overall microcapillary network occlusion, was quantified. SCD Biochip microchannels were fabricated by lamination and were functionalized with human laminin (LN-511) [2]. Undiluted whole blood was injected into the microchannel at 1 dyne/cm 2, a shear stress value typically observed in the post-capillary venules. Following a wash step, the SCD Biochip microchannel was imaged, and the number of adherent RBCs in a 32-mm 2 window was quantified. For hypoxia experiments, a hypoxic setup was fabricated for blood deoxygenation (pO 2 ~45 mmHg) [3, 4]. Ektacytometry measurements were performed according to the manufacturers' specifications (Lorrca Maxsis). Data are reported as mean ± standard deviation (SD). Results: We initially analyzed RBC-mediated microvascular occlusion under normoxia or hypoxia using the OcclusionChip (Figure 1A). Under normoxia, HbSS-containing RBCs had relatively greater OI values compared to HbAA- and HbAS-containing RBCs (Figure 1B, P = 0.057 for HbSS vs HbAA and P = 0.060 for HbSS vs HbAS). However, exposure to hypoxia led to significantly elevated OI values in the HbAS- and HbSS-containing RBCs (Figure 1B, 0.05 ± 0.02% vs 33.62 ± 18.31%, P = 0.015 for HbAS, and 0.27 ± 0.24% vs 49.37 ± 24.47%, P = 0.001 for HbSS, normoxia vs hypoxia). Negligible occlusion was observed in HbAA-containing RBCs (Figure 1B). We then analyzed RBC adhesion to LN under normoxia or hypoxia using the SCD Biochip (Figure 1C). Hypoxia led to greater number of adherent RBCs on LN in the HbSS-containing RBCs (Figure 1D, 141 ± 91 vs 497 ± 392, P = 0.089, normoxia vs hypoxia), but this effect was not present in HbAA- or HbAS-containing RBCs (Figure 1B, 2 ± 1 vs 3 ± 1, P > 0.05 for HbAA, and 10 ± 7 vs 12 ± 3, P > 0.05 for HbAS, normoxia vs hypoxia). Further, under normoxia, we found that the HbAS-containing RBCs had slightly greater number of adherent RBCs on LN compared to the HbAA-containing RBCs (Figure 1D, P = 0.057 for HbAA vs HbAS). As previously reported, HbSS-containing RBCs showed greatest adhesion to LN under normoxia compared to the HbAA- and HbAS-containing RBCs (Figure 1D, P = 0.027 for HbSS vs HbAA and P = 0.033 for HbSS vs HbAS)., Finally, we preformed Lorrca oxyscan and found that ektacytometry is less sensitive to RBC deformability change under hypoxia in SCT (Figure 1E). Conclusions: Findings in this study suggest that although RBCs from subjects with SCT are deformable under normoxia and are able to clear narrow capillaries similar to normal RBCs, hypoxia changes deformability, presumably due to hypoxic polymer formation, and could contribute to microvascular occlusion in SCT. The OcclusionChip is a single cell-based technology, and may be more sensitive to single RBC deformability. Future studies will prospectively focus on analyzing RBC adhesion on activated microvascular endothelial cells in physiologic flow to further interrogate the impact of hypoxia on pathophysiology in SCT. References: [1] Man et al., LabChip, 2020, 20, 2086-2099. [2] Kim et al., Microcirculation, 2017, 24, e12374. Figure 1 Figure 1. Disclosures An: Hemex Health, Inc.: Patents & Royalties. Kucukal: BioChip Labs: Current Employment, Patents & Royalties. Nayak: BioChip Labs: Patents & Royalties. Little: Biochip Labs: Patents & Royalties; Hemex Health, Inc.: Patents & Royalties. Gurkan: Dx Now Inc.: Patents & Royalties; Hemex Health, Inc.: Current Employment, Patents & Royalties; Biochip Labs: Patents & Royalties; Xatek Inc.: Patents & Royalties.

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The acceleration of the propagation phase of thrombin generation in patients with steady-state sickle cell disease is associated with circulating erythrocyte-derived microparticles

Thrombosis and Haemostasis ◽

10.1160/th11-10-0689 ◽

2012 ◽

Vol 107 (06) ◽

pp. 1044-1052 ◽

Cited By ~ 44

Author(s):

Grigoris Gerotziafas ◽

Patrick Van Dreden ◽

Mourad Chaari ◽

Vassiliki Galea ◽

Amir Khaterchi ◽

...

Keyword(s):

Steady State ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Thrombin Generation ◽

Generation Process ◽

Cell Disease ◽

Significant Acceleration ◽

High Concentrations ◽

The Impact ◽

Functional Alteration

SummarySickle cell disease (SCD) is linked to hypercoagulability and is characterised by high concentrations of erythrocyte-derived microparticles (Ed-MPs). However, the impact of procoagulant cell-derived microparticles on the thrombin generation process remains unclear. We analysed the alterations of each phase of thrombin generation (TG) in relation to the concentration of erythrocyte- or platelet-derived microparticles (Ed-MPs and Pd-MPs) in a cohort of patients with steady-state SCD. We studied 92 steady-state SCD patients, 19 of which were under treatment with hydroxyurea, and 30 healthy age- and sex-matched individuals. TG was assessed by calibrated automated thrombogram. Ed-MP and Pd-MP expressing or not phosphatidylserine (PS) were determined by means of flow cytometry. Procoagulant phospholipid-dependent activity in the plasma was evaluated by the Procoag-PPL assay. Levels of thrombomodulin and haemoglobin in the plasma as well as red blood cell and reticulocyte counts were measured. SCD patients, independently of the administration of hydroxyurea, were marked by a significant acceleration in the propagation phase of TG which correlated with the Ed-MP/PS+ concentration. TG was significantly attenuated in hydroxyurea-treated patients. In conclusion, the acceleration of the propagation phase of TG, driven by Ed-MP/PS+, is a major functional alteration in blood coagulation in patients with steady-state SCD. Treatment with hydroxyurea, in addition to the regulation of haemolysis, lowers Ed-MPs and attenuates thrombin generation. The thrombogram could be a useful tool for the diagnosis of hypercoagulability and optimisation of the treatment in patients with SCD.

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Persistence of Chronic Inflammation Despite Years of Transfusion Program in SCD Patients: Changing Red Blood Cells Is Not Sufficient to Treat Sickle Cell Disease

Blood ◽

10.1182/blood-2021-147787 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 764-764

Author(s):

Abdoul Karim Dembele ◽

Patricia Hermand-Tournamille ◽

Florence Missud ◽

Emmanuelle Lesprit ◽

Malika Benkerrou ◽

...

Keyword(s):

Endothelial Cells ◽

Sickle Cell Disease ◽

Red Blood Cells ◽

Chronic Inflammation ◽

Sickle Cell ◽

Blood Cells ◽

Healthy Controls ◽

Cell Disease ◽

The Impact

Abstract Sickle cell disease (SCD) is a severe hemoglobinopathy due to abnormal hemoglobin S (HbS). Although red blood cell dysfunction is at the core of the SCD pathophysiology, several studies have highlighted the important role of inflammatory cells like neutrophils. One of the most serious complications of SCD is cerebral vasculopathy (CV), due to the occlusion of one or more intracranial or cervical arteries. In 1998, the STOP study demonstrated that monthly blood transfusions could reduce the risk of stroke by 90% in children with CV. However, there is large heterogeneity in the evolution of CV under chronic transfusion, sometimes requiring exchange transfusion (ET) program for years without succeeding in healing the CV. The aim of the study is to investigate the impact of long-term transfusion program on neutrophil dysfunction, in order to understand if persistent inflammation could contribute to the non-healing of CV despite HbS permanently below 40%. In SCD children undergoing ET program for at least 1 year, we analysed i)the phenotype of neutrophils with 8 markers of activation/adhesion/ageing, ii)the plasmatic levels of elastase, witnessing the NETose activity of neutrophils, and iii)the ex-vivo adhesion of neutrophils on activated endothelial cells. One hundred and two SCD children with an ET transfusion program for at least 6 months because of CV were included in the study. ET session, carried out every 5 weeks and most of the time by erythrapheresis, reached their biological objectives with a mean HbS rate after ET session of 14.1%, and 35.4% before the next ET session, which means that these patients globally live at an average HbS level of 24% for at least 1 year. We managed to limit iron overload with a mean ferritinemia of 207 µg/L in the whole cohort. Despite these satisfactory results in terms of HbS reduction, the efficiency in curing the CV was modest in accordance with the previously described efficiency of ET program in SCD children: after a mean ET program duration of 4.4 years only 22% of them had an improvement of their CV since the beginning of the ET program, while 60% of them had a stagnation of their CV, and 18% of them worsened their vascular lesions. Considering inflammatory parameters, the patients had persistence of high leukocytosis and high neutrophils count (respective mean of 9810 G/L and 5742 G/L), significantly not different of neutrophils count before inclusion in the ET program. In a random subgroup of 20 patients, we analysed neutrophils phenotype, NETose and endothelial adhesion and compared them to healthy controls and SCD children without ET, treated or not with Hydroxyurea (HU). Overall, we observed as expected an activated, aged and adherent profile of neutrophils from untreated SCD children compared to healthy controls, characterized by an overexpression of CD18/CD11b (p=0,03), CD18/CD11a (p=0,02), CD162 (p=0,01), CD66a (p=0,01) and the ageing markers CD184 high/CD62Llow (p=0,04) as well as a higher plasmatic level of elastase (p=0. 01) and higher adhesion of neutrophils to endothelial cells. All these parameters were alleviated in SCD patients treated with HU. In SCD patient undergoing ET program, we found a similar profile of activated neutrophils to that of untreated SCD patients with a similar expression of activation molecules, high level of elastase and the same increase of neutrophils adhesion to endothelial cells compared to controls, witnessing a persistence of chronic inflammation despites years of ET. Overall, our study highlights that the replacement of sickle red blood cells, even for years, is not sufficient to reverse the deleterious inflammatory phenotype of neutrophils. Given the major role of inflammation in endothelial dysfunction, these could contribute to the persistence of CV in a majority of patients despite efficient ET programs. This raises the question of systematically combining ET program with anti-inflammatory treatment such as HU or P-selectin inhibitors in children with CV. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Sickle cell disease promotes sex-dependent pathological bone loss through enhanced cathepsin proteolytic activity

Blood Advances ◽

10.1182/bloodadvances.2021004615 ◽

2021 ◽

Author(s):

Jada M Selma ◽

Hannah Song ◽

Christian Rivera ◽

Simone Andrea Douglas ◽

Abhiramgopal Akella ◽

...

Keyword(s):

Sickle Cell Disease ◽

Bone Loss ◽

Sickle Cell ◽

Sickle Cell Trait ◽

Bone Microarchitecture ◽

The United States ◽

Cell Disease ◽

Female Mice ◽

Blood Disorder ◽

The Impact

Sickle cell disease (SCD) is the most common hereditary blood disorder in the United States. SCD is frequently associated with osteonecrosis, osteoporosis and osteopenia and other bone related complications such as vaso-occlusive pain, ischemic damage, osteomyelitis, and bone marrow hyperplasia known as sickle bone disease (SBD)1,2. Previous SBD models have failed to distinguish the age- and sex-specific characteristics of bone morphometry. In this study, we use the Townes mouse model of SCD to study the pathophysiological complications of SBD in both SCD and sickle cell trait. Changes in bone microarchitecture and bone development were assessed by high-resolution quantitative micro-computed tomography (microCT) and the 3D reconstruction of femurs from male and female mice. Our results indicate that SCD causes bone loss and sex-dependent anatomical changes in bone. Particularly, SCD female mice are prone to trabecular bone loss while cortical bone degradation occurs in both sexes. Additionally, we describe the impact of genetic knockdown of cathepsin K and E-64 mediated cathepsin inhibition on SBD.

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

Blood ◽

10.1182/blood-2020-142935 ◽

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< 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<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<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.

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

Blood ◽

10.1182/blood-2018-99-118440 ◽

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.

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

Blood ◽

10.1182/blood-2002-11-3422 ◽

2003 ◽

Vol 102 (4) ◽

pp. 1506-1514 ◽

Cited By ~ 104

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 < .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 < .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 < .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.

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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 ◽

10.1182/blood-2019-126371 ◽

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: > 75th percentile, 455.9 cells/mm2; mild/moderate adhesion phenotype: < 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<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.

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