scholarly journals Neutrophil-Platelet Aggregation Enables Vaso-Occlusion in Sickle Cell Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1295-1295 ◽  
Author(s):  
Maritza A. Jimenez ◽  
Gregory J Kato ◽  
Prithu Sundd
Keyword(s):  
Platelet Aggregation ◽  
Sickle Cell Disease ◽  
Fluorescence Microscopy ◽  
Single Cell ◽  
Sickle Cell ◽  
Genetic Disorder ◽  
Cellular Interactions ◽  
Cell Disease ◽  
Microfluidic Flow ◽  
Neutrophil Aggregation

Introduction: Sickle Cell Disease (SCD) is an autosomal-recessive-genetic disorder that leads to sickling and hemolysis of red blood cells (RBCs). Acute vaso-occlusive pain crisis (VOC) is the predominant pathophysiology faced by SCD patients and the primary reason for emergency medical care. Although neutrophils have been shown to play a role in vaso-occlusion by interacting with sickle RBCs in the cremaster venules of transgenic SCD mice, the cellular, molecular and biophysical mechanisms that promote vaso-occlusion in SS patients is not completely understood. Materials and Methods: Freshly collected heparinized blood from steady-state SS patients and race matched control (AA) subjects was perfused through silicone based microfluidic flow channels with a glass bottom coated a cocktail of recombinant human P-selectin, ICAM-1 and IL-8 at a physiological wall 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 at a single cell-resolution using quantitative microfluidic fluorescence microscopy (qMFM)1, which allows quantitative assessment of vaso-occlusive events at an unprecedented single cell resolution2. Results: Vaso-occlusion in the microfluidic channel involved neutrophil arrest followed by nucleation of platelets on arrested neutrophils, formation of neutrophil-platelet-aggregates (NPA) and partial occlusion of the microfluidic flow channel. Remarkably, the number of platelet-neutrophil interactions and the lifetime of these interactions were several folds higher in SS patient than control AA blood. Surprisingly, preincubation with 250 ng/ml of bacterial lipopolysaccharide (LPS) led to a significant increase in the number and lifetime of platelet-neutrophil interactions in SS but not AA blood. This enhanced NPA formation in SS patient blood was attenuated to the level observed in AA blood by simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils as well as pretreatment with a small molecule inhibitor of toll-like-receptor-4 (TLR4) signaling pathway. Conclusion: Our data shows that the vaso-occlusive pathophysiology in SCD involves sequential steps of neutrophil arrest, nucleation of platelets on arrested neutrophils, formation of large NPAs and obstruction of blood flow. Platelet-neutrophil aggregation can be ameliorated by the simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils. The inflammatory milieu of SS patient blood sets a lower threshold for bacterial endotoxin induced neutrophil-platelet aggregation than control blood. The enhanced platelet-neutrophil aggregation in SS blood is dependent on activation of TLR-4 pathway. Understanding the molecular mechanism of vaso-occlusion will enable the development of therapeutics to 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;100(10):e390-e393. doi:10.3324/haematol.2015.126631. 2 Sundd, P. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nat Methods7, 821-824, doi:10.1038/nmeth.1508 (2010). Disclosures Kato: Mast Therapeutics: Consultancy; Bayer: Research Funding.

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 Targeting pain at its source in sickle cell disease

2018 ◽  
Vol 315 (1) ◽  
pp. R104-R112 ◽  
Author(s):  
Kanika Gupta ◽  
Om Jahagirdar ◽  
Kalpna Gupta
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
Genetic Disorder ◽  
Somatosensory System ◽  
Organ Damage ◽  
Mast Cell Activation ◽  
Cell Disease ◽  
Central Nervous Systems

Sickle cell disease (SCD) is a genetic disorder associated with hemolytic anemia, end-organ damage, reduced survival, and pain. One of the unique features of SCD is recurrent and unpredictable episodes of acute pain due to vasoocclusive crisis requiring hospitalization. Additionally, patients with SCD often develop chronic persistent pain. Currently, sickle cell pain is treated with opioids, an approach limited by adverse effects. Because pain can start at infancy and continue throughout life, preventing the genesis of pain may be relatively better than treating the pain once it has been evoked. Therefore, we provide insights into the cellular and molecular mechanisms of sickle cell pain that contribute to the activation of the somatosensory system in the peripheral and central nervous systems. These mechanisms include mast cell activation and neurogenic inflammation, peripheral nociceptor sensitization, maladaptation of spinal signals, central sensitization, and modulation of neural circuits in the brain. In this review, we describe potential preventive/therapeutic targets and their targeting with novel pharmacologic and/or integrative approaches to ameliorate sickle cell pain.

scholarly journals Prevalence of Sickle Cell Disease and Other Haemoglobin Variants in Calabar, Cross River State, Nigeria

2019 ◽  
pp. 1-6
Author(s):  
Akaba Kingsley ◽  
Ofem Enang ◽  
Ofonime Essien ◽  
Annette Legogie ◽  
Omini Cletus ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Genetic Disorder ◽  
Globin Gene ◽  
Specific Gene ◽  
Cell Disease ◽  
Cellulose Acetate Electrophoresis ◽  
Genetic Changes ◽  
Female Ratio ◽  
Cross River State

Background: Sickle cell disease (SCD) is the commonest genetic disorder worldwide with a global prevalence of 20-25 million. About 12-15 million affected persons are in Sub-Sahara Africa with Nigeria bearing the highest burden of people living with sickle cell disease. SCD is a disease characterized as an autosomal, recessive, heterogeneous, and a monogenetic disorder caused by an A-to-T point mutation in the β-globin gene responsible for the production of abnormal hemoglobin S (HbS), which polymerizes in the deoxygenated state and results in the sickling of erythrocytes.  Haemoglobin variants are mutant forms of haemoglobin in a population usually occurring as a result of genetic changes in specific genes, or globins that causes change on alterations in the amino acid. They could affect the structure, behavior, the production rate and the stability of the specific gene. Well-known haemoglobin variants such as sick-cell anaemia are responsible for diseases and are considered haemoglobinopathies. Other variants cause no detectable pathology and are thus considered as non-pathological variants. Aim: The study is aimed at evaluating the burden of sickle cell disease and other haemoglobin variants in Calabar, South-South Nigeria. Methods: This is a retrospective study done at the haematology laboratory of University of Calabar Teaching Hospital, Calabar. Cellulose acetate electrophoresis at alkaline pH was used for the evaluation of haemoglobinopathies. The data were entered into Microsoft Excel 2016 spreadsheet and analysed with the IBM SPSS Version 22. Data were summarized into percentage of different phenotypes. Results: Results of the total 3648 haemoglobin electrophoresis recorded, 1368 (37.50%) were male while the remaining 2280 (62.5%) females given a male to female ratio of 1:1.7. Five haemoglobin phenotypes were identified as HbAA, HbAS, HbAC, HbSC and HbSS. The overall average values of their prevalence were HbAA 64.78%, HbAS 32.62%, HbSS 2.14%, HbAC 0.33%, HbSC 0.14%. Thus, the prevalence of SCD (Prevalence of HbSS+HbSC) was 2.28%. The highest proportion of SCD was observed in 2011 with least in 2016 and 2017 respectively. Conclusion: The prevalence of SCD and other haemoglobin variants in Calabar is similar to that of the national prevalence rate. There is need for continuous enlightenment and premarital counselling on the pattern of inheritance of SCD most especially with the increased burden of sickle traits in the environment has reported in this study.

scholarly journals Sickle Cell Disease: An Overview of Oral Health Considerations for Pediatric Patients

2021 ◽  
Vol 2 (3) ◽  
pp. 9-17
Author(s):  
Dafni Eleftherou ◽  
Aristidis Arhakis ◽  
Sotiria Davidopoulou
Keyword(s):  
Oral Health ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Pediatric Patients ◽  
Genetic Disorder ◽  
The Body ◽  
Common Finding ◽  
Cell Disease ◽  
Preventive Dental Care ◽  
Periodontal Inflammation

Aim: This literature review aims to update the evidence for orofacial manifestations and current treatment recommendations for children and adolescents with sickle cell disease. Background: Sickle cell disease is a frequent hemoglobinopathy and a life-threatening genetic disorder. The lifelong condition is characterized by chronic hemolytic anemia and vaso-occlusive crisis that may occur in a variable range of clinical presentations in different regions of the body, including the oral cavity. Review results: This review explored the most common orofacial alterations of pediatric patients with SCD. Dental caries is a common finding in SCD pediatric patients, especially in those who are socio-economically vulnerable. Moreover, malocclusions occur in high prevalence in SCD pediatric patients. Other oral health complications seen in SCD patients include periodontal inflammation, bone changes, infections, mental nerve neuropathy, facial overgrowth, delayed tooth eruption, dental anomalies, pulp necrosis, soft tissue alterations and salivary changes. Dental infections may trigger a vaso-occlusive crisis leading the patient to a higher probability on arriving in hospital emergency departments and in need for further hospital admission to deal with the correlated complications. Thus, preventive dental care and non-invasive dental procedures are the principal focus in SCD patients in order to avoid possible subsequent complications. Conclusion: The review showed that in pediatric patients with SCD the risk for orofacial manifestations and complications depends not only on the presence of SCD but also on other confounding factors such as oral hygiene, diet habits and social conditions. Moreover, more well-designed epidemiological studies are necessary to assess the real link between SCD disease and its impact on stomatognathic health.

scholarly journals Zinc Finger Nuclease-Mediated Disruption of the BCL11A Erythroid Enhancer Results in Enriched Biallelic Editing, Increased Fetal Hemoglobin, and Reduced Sickling in Erythroid Cells Derived from Sickle Cell Disease Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 974-974 ◽  
Author(s):  
Samuel Lessard ◽  
Pauline Rimmele ◽  
Hui Ling ◽  
Kevin Moran ◽  
Benjamin Vieira ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Cell Therapy ◽  
Single Cell ◽  
Zinc Finger ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Potential Efficacy

High fetal hemoglobin (HbF) levels are associated with decreased severity and mortality in sickle cell disease (SCD) and beta thalassemia (BT). We have developed a novel gene-edited cell therapy using autologous hematopoietic stem and progenitor cells (HSPCs) that have been genetically modified with zinc finger nucleases (ZFNs) to reactivate HbF expression. The ZFNs target the binding motif of GATA1 (GATAA) within an intronic erythroid-specific enhancer (ESE) of BCL11A, which encodes a major transcriptional repressor of HbF. Previously, we reported successful ZFN-mediated editing of the BCL11A ESE and reactivation of HbF in both dual (granulocyte colony-stimulating factor (G-CSF) and plerixafor) and single plerixafor mobilized HSPCs(Holmes 2017, Moran 2018). Both related drug candidates, ST-400 and BIVV003, are currently in phase 1/2a clinical trials for transfusion-dependent BT (NCT03432364) and SCD (NCT03653247), respectively. Here, we performed extensive genetic and phenotypic characterization of ZFN-edited HSPCs from healthy and SCD donors. We performed single-cell characterization of BCL11A ESE-edited HSPCs from 4 healthy donors. Briefly, individual HSPCs were sorted and cultured in erythroid differentiation medium. Genomic DNA and protein lysate were collected at day 14 and 20, respectively. In total, we successfully genotyped 961 single-cell derived colonies by next-generation sequencing. The distribution was highly skewed towards biallelic-edited cells (P<3x10-149) representing 94% of edited clones, suggesting that ZFN-expressing cells are likely to become edited at both alleles. We found that each edited allele contributed additively to an increase in HbF% of 15% (P=1x10-80) as measured by UPLC. Clones harboring GATAA-disrupting indels on both alleles displayed on average 34% more HbF% than WT clones (P=1x10-112). In contrast, clones with biallelic indels that left the motif intact displayed a more modest increase (13%, P=1x10-6). Overall, our data revealed that >90% of edited cells were biallelic, displaying on average 27-38% more HbF% despite variation in donor baseline levels. We observed a strong enrichment of biallelic-edited homozygotes (same indel pattern at both alleles) compared to an expected random distribution (161 vs 24; P<1x10-5). These clones may harbor larger deletions not captured by sequencing, as reported previously using CRISPR/Cas9 (Kosicki 2018). To address this question, we used a combination of a small amplicon sequencing assay design covering an informative SNP and a 12kb amplicon Nextera assay. We found that 27% of initially assigned homozygote clones were bona fide homozygotes (44/161) with the remaining harboring indels not originally captured. Nevertheless, most indels remained small, with 91% of indels <50bp, and deletions and insertions >1kb together consisting of less than 1% of alleles. The largest deletion was 4kb, but no indel extended outside the enhancer region of BCL11A or altered the coding region (>26 kb away). Moreover indels >50bp were not associated with enucleation levels (P=0.77), suggesting that they did not alter erythroid function. Overall, these results are consistent with previous data showing that ZFN-mediated gene editing does not impair HSPC function in vitro based on colony forming unit (CFU) production, and that injection of BIVV003 into immune-deficient NBSGW mice results in robust long-term engraftment with no impact on the number of HSPCs or their progeny, including erythrocytes. Finally, BCL11A ESE editing in HSPCs mobilized from one SCD donor resulted in a 3-fold HbF increase consistent across technical duplicates, without impacting CFU production or erythroid enucleation. Importantly, clonal analysis revealed a similar enrichment of biallelic editing (P=6x10-4) and additive HbF up-regulation, with biallelic edited cells reaching 28% more HbF% than unedited cells (50% vs 22%, P=7x10-5). Furthermore, enucleated cells differentiated from edited HSPCs showed attenuation of sickling under hypoxic conditions supporting the potential efficacy of BIVV003. Experiments in HSPCs from additional SCD donors are ongoing. Overall, our data have shown that ZFN-mediated disruption of BCL11A ESE results in enriched biallelic editing with on-target small indels, reactivates HbF and reduces sickling, supporting the potential efficacy and specificity of BIVV003 as a novel cell therapy for SCD. Disclosures Lessard: Sanofi: Employment. Rimmele:Sanofi: Employment. Ling:Sanofi: Employment. Moran:Sanofi: Employment. Vieira:Sanofi: Employment. Lin:Sanofi: Employment. Hong:Sanofi: Employment. Reik:Sangamo Therapeutics: Employment. Dang:Sangamo Therapeutics: Employment. Rendo:Sanofi: Employment. Daak:Sanofi: Employment. Hicks:Sanofi: Employment.

ABNORMALITIES OF PLATELET AGGREGATION AND ENHANCED FACTOR X ACTIVATOR ACTIVITY OF WASHED PLATELETS IN SICKLE CELL DISEASE

1987 ◽  
Author(s):  
D A F Chamone ◽  
A Y Hoshikawa-Fujimura ◽  
C Massumoto ◽  
G Bellotti ◽  
F Arashiro ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Vascular Endothelium ◽  
Platelet Rich Plasma ◽  
Coagulation Factors ◽  
Factor X ◽  
Cell Disease ◽  
Normal Volunteers ◽  
Pathologic Features

The occurence of microvascular occlusion is one of the most prominent pathologic features of sickle cell anemia. The mechanism of vaso occlusion has generally been attributed to the abnormal shape and reduced deformability of the sickled erithrocy tes. However, the involvement of vascular endothelium, platelets and their interactions with coagulation factors may also be of pathogenic significance in microvascular occlusive crises.We investigated the interaction between vascular endothelium, platelets and blood coagulation factors in 23 patients with Sickle Cell Disease (SCD) and in normal volunteers.Factor X activator activity in washed platelets was performed according to Semeraro and Vermylen (1977), thromboxane B2 (TXB2) and 6-keto-PGF1β were determined using specific radioimmunoassays.. PAF-acether from platelets was determined according to Chignard et al (Nature, 1979, 279:799). Platelet aggregation was performed with a Chrono-Log Aggregometer (Model 440) on platelet rich plasma (PRP) using the Born method. Prostacyclin release from endothelium was performed according to Mon-cada et al (Lancet i:18, 1977).Our results showed that platelets from patients with SCD ha ve enhanced factor X activator activity (p < 0.0001), produce mo re PAF-acether than controls (p < 0.02) and showed hyperaggregability in these patients as compared to normal volunteers (p < 0.00001).We concluded that platelets from homozygous sicklers have enhanced factor X activator activity as well as increased capacity for PAF-acether production. These abnormalities may contribute to the incidence of vaso occlusive crises in these patients.

scholarly journals Sickle Cell Disease: Management options and challenges in developing countries

2013 ◽  
Vol 5 (1) ◽  
pp. e2013062 ◽  
Author(s):  
Daniel Ansong ◽  
Alex Osei-Akoto ◽  
Delaena Ocloo ◽  
Kwaku Ohene-Frempong Ohene-Frempong
Keyword(s):  
Developing Countries ◽  
Sickle Cell Disease ◽  
Newborn Screening ◽  
Sickle Cell ◽  
Health Care Professionals ◽  
Genetic Disorder ◽  
Sub Saharan Africa ◽  
Acute Chest Syndrome ◽  
Cell Disease ◽  
Management Options

Sickle Cell Disease (SCD) is the most common genetic disorder of haemoglobin in sub-Saharan Africa. This commentary focuses on the management options available and the challenges that health care professionals in developing countries face in caring for patients with SCD. In developing countries like Ghana, newborn screening is now being implemented on a national scale.  Common and important morbidities associated with SCD are vaso-occlusive episodes, infections, Acute Chest Syndrome (ACS), Stroke and hip necrosis. Approaches to the management of these morbidities are far advanced in the developed countries. The differences in setting and resource limitations in developing countries bring challenges that have a major influence in management options in developing countries. Obviously clinicians in developing countries face challenges in managing SCD patients. However understanding the disease, its progression, and instituting the appropriate preventive methods are paramount in its management. Emphasis should be placed on newborn screening, anti-microbial prophylaxis, vaccination against infections, and training of healthcare workers, patients and caregivers. These interventions are affordable in developing countries.

Multicenter Evaluation of HemoTypeSC as a Point-of-Care Sickle Cell Disease Rapid Diagnostic Test for Newborns and Adults Across India

2019 ◽  
Vol 153 (1) ◽  
pp. 82-87 ◽  
Author(s):  
Malay B Mukherjee ◽  
Roshan B Colah ◽  
Pallavi R Mehta ◽  
Nikhil Shinde ◽  
Dipty Jain ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Newborn Screening ◽  
Sickle Cell ◽  
High Performance ◽  
Screening Program ◽  
Point Of Care ◽  
Genetic Disorder ◽  
Cell Disease ◽  
Point Of Care Test ◽  
Tribal Areas

Abstract Objectives Sickle cell anemia is the commonest genetic disorder in India, and the frequency of the sickle cell gene is very high in the remote tribal areas where facilities are generally limited. Therefore, a rapid and affordable point-of-care test for sickle cell disease is needed. Methods The diagnostic accuracy of HemoTypeSC was evaluated against automated high-performance liquid chromatography (HPLC) as the gold standard for its efficacy in a newborn screening program. Results A total of 1,559 individuals (980 newborns and 579 adults) from four participating centers were analyzed by both methods. HemoTypeSC correctly identified 209 of 211 total hemoglobin (Hb) SS cases, for a 99.1%/99.9% total HbSS sensitivity/specificity. Overall, HemoTypeSC exhibited sensitivity and specificity of 98.1% and 99.1% for all possible phenotypes (HbAA, HbAS, and HbSS) detected. HPLC is relatively expensive and not available in most laboratories in remote tribal areas. Conclusions We conclude that the rapid, point-of-care testing device HemoTypeSC test is suitable for population and newborn screening for the HbS phenotype.

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.

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