Red Blood Cell Rigidity Research Articles

Visualization and Measurements of Blood Cells Flowing in Microfluidic Systems and Blood Rheology: A Personalized Medicine Perspective.

Hemorheological alterations in the majority of metabolic diseases are always connected with blood rheology disturbances, such as the increase of blood and plasma viscosity, cell aggregation enhancement, and reduction of the red blood cells (RBCs) deformability. Thus, the visualizations and measurements of blood cells deformability flowing in microfluidic devices (point-of-care devices) can provide vital information to diagnose early symptoms of blood diseases and consequently to be used as a fast clinical tool for early detection of biomarkers. For instance, RBCs rigidity has been correlated with myocardial infarction, diabetes mellitus, hypertension, among other blood diseases. In order to better understand the blood cells behavior in microfluidic devices, rheological properties analysis is gaining interest by the biomedical committee, since it is strongly dependent on the interactions and mechanical cells proprieties. In addition, the development of blood analogue fluids capable of reproducing the rheological properties of blood and mimic the RBCs behavior at in vitro conditions is crucial for the design, performance and optimization of the microfluidic devices frequently used for personalized medicine. By combining the unique features of the hemorheology and microfluidic technology for single-cell analysis, valuable advances in personalized medicine for new treatments and diagnosis approach can be achieved.

Neuroimaging in Sickle Cell Disease: A Review.

Sickle cell disease is the most common hereditary hemoglobinopathy, which results in abnormally shaped and rigid red blood cells. These sickle-shaped red blood cells cause vaso-occlusion and ischemic phenomena that can affect any organ in the body. As a common cause of disability, the neurological manifestations of sickle cell disease are particularly important. Neuroimaging has a crucial role in the diagnosis, management, and prevention of the complications of sickle cell disease. These complications can affect the brain parenchyma, vasculature, and skull and can be ascribed directly or indirectly to a vasculopathy of small and large vessels. Vaso-occlusion can cause ischemic stroke. Ischemic damage in the absence of an acute neurological deficit, and therefore only apparent on neuroimaging, is termed silent cerebral ischemia. Weakening of the arterial walls can cause aneurysms. In its most severe form, a vasculopathy of the terminal internal carotid arteries can progress to moyamoya syndrome, characterized by steno-occlusive disease and the formation of friable collateral arteries. Rupture of aneurysms or friable collateral arteries is a potential cause of intracranial hemorrhage. The skull and vertebrae may be affected by extra-medullary hematopoiesis, due to severe anemia, or iron deposition, due to chronic red blood cell transfusion. Impaired blood supply to bone is associated with osteomyelitis and osteonecrosis. Fat embolization syndrome is a rare complication of osteonecrosis, which may cause devastating neurological impairment. Awareness and early recognition of the diverse manifestations of sickle cell disease on neuroimaging is crucial to ensure optimal treatment in a complex patient cohort.

Aberrant Membrane Composition and Biophysical Properties Impair Erythrocyte Morphology and Functionality in Elliptocytosis.

Red blood cell (RBC) deformability is altered in inherited RBC disorders but the mechanism behind this is poorly understood. Here, we explored the molecular, biophysical, morphological, and functional consequences of α-spectrin mutations in a patient with hereditary elliptocytosis (pEl) almost exclusively expressing the Pro260 variant of SPTA1 and her mother (pElm), heterozygous for this mutation. At the molecular level, the pEI RBC proteome was globally preserved but spectrin density at cell edges was increased. Decreased phosphatidylserine vs. increased lysophosphatidylserine species, and enhanced lipid peroxidation, methemoglobin, and plasma acid sphingomyelinase (aSMase) activity were observed. At the biophysical level, although membrane transversal asymmetry was preserved, curvature at RBC edges and rigidity were increased. Lipid domains were altered for membrane:cytoskeleton anchorage, cholesterol content and response to Ca2+ exchange stimulation. At the morphological and functional levels, pEl RBCs exhibited reduced size and circularity, increased fragility and impaired membrane Ca2+ exchanges. The contribution of increased membrane curvature to the pEl phenotype was shown by mechanistic experiments in healthy RBCs upon lysophosphatidylserine membrane insertion. The role of lipid domain defects was proved by cholesterol depletion and aSMase inhibition in pEl. The data indicate that aberrant membrane content and biophysical properties alter pEl RBC morphology and functionality.

MON-LB127 Pulmonary Embolism in the Setting of Diabetic Ketoacidosis. an Under-Recognized Complication

The relation between Diabetic Ketoacidosis and Venous Thromboembolism is important to appreciate for early recognition and management. 90 year old female with Type 2 DM was brought to the ER after a syncopal episode. Family reported the patient to be lethargic with a decreased appetite for one week. On arrival, patient had a blood pressure of 84/44 mmHg. Oxygen saturation was 89% at room air and improved to 93% on supplemental O2. Patient was afebrile with a respiratory rate of 16 and heart rate 89/minute. On exam, she was dehydrated with decreased skin turgor, dry oral mucosa. Labs revealed blood glucose of 621 mg/dL, Bicarbonate 16 mmol/L, B-Hydroxybutyrate 5.00 mmol/L, Anion Gap 21, pH of 7.26 on ABG. Urinalysis was suggestive of a urinary tract infection. After initiation of IV antibiotics and insulin, she was transferred to the intensive care unit. In the ICU, her blood pressure failed to improve with fluid resuscitation, ultimately requiring vasopressors. Due to hypotension with hypoxia, CT Chest was performed which revealed extensive bilateral PE. She was started on IV heparin infusion. Pro-Brain Natriuretic Peptide was elevated at 4,716 pg/mL. Echocardiogram confirmed right heart strain with severely dilated right ventricle, positive McConnell’s sign, systolic and diastolic septal flattening and an estimated RSVP 67mmHg consistent with moderately severe pulmonary hypertension. Tissue plasminogen activator was recommended however given the patient’s age and functional status; family decided against systemic thrombolysis. Duplex ultrasound of her lower extremities also showed bilateral acute deep venous thrombosis. She was continued on intravenous anticoagulation and eventually was able to come off vasopressors. As the patient’s blood glucose levels improved and her anion gap closed, she was transitioned to basal/bolus insulin and transferred to the general medical floor. She was started on Apixaban and discharged home per family’s request after her code status was changed to DNR-Comfort Care Arrest.Pulmonary embolism is a serious venous thromboembolic event that is rarely reported in association with DKA. Proposed mechanisms include some of the same mechanisms implicated in arterial thrombosis, namely abnormalities in coagulation factors, increased platelet aggregation, impaired fibrinolysis and endothelial injury due to hypertonicity. Also, severe dehydration associated with DKA may contribute by virtue of increased red blood cell rigidity and increased blood viscosity establishing DKA as an underlying cause or contributing factor of pulmonary thromboembolism2.1 Langevin C et al Presumed paradoxical embolus in a patient with diabetic ketoacidosis Int J Gen Med 2015;8:297–301 2015 Sep 182 Scordi Bello I et al Fatal Pulmonary Thromboembolism in Patients with Diabetic Ketoacidosis A Seven-Case Series and Review of the Literature Acad Forensic Pathol 2016;6(2):198–205

An experimental erythrocyte rigidity index (Ri) and its correlations with Transcranial Doppler velocities (TAMMV), Gosling Pulsatility Index PI, hematocrit, hemoglobin concentration and red cell distribution width (RDW).

Brain artery velocities (Time-Averaged Maximum Mean Velocity, TAMMV) by Transcranial Doppler (TCD), hematocrit, hemoglobin, Red blood cell (RBC) Distribution Width (RDW) and RBC rigidity index (Ri), when reported together with their correlations, provide a accurate and useful diagnostic picture than blood viscosity measurements alone. Additionally, our study included a sixth parameter provided by TCD, the Gosling Pulsatility Index PI, which is an indicator of CBF (Cerebral Blood Flow) resistance. All these parameters are routine in Hematology except for values of Ri. The rigidity (Ri) of the RBC is the main rheological characteristic of the blood of Sickle Cell Anemia (SCA) patients and several pathologies. However, its quantification depends on many commercial and experimental techniques, none disseminated and predominant around the World. The difference in absorbance values of the blood, during the process of sedimentation in a microwell of a Microplate Reader, is a straightforward way of semi-quantifying the RBC rigidity Ri, since the fraction of irreversibly sickled red blood cells does not form rouleaux. Erythrocyte Rigidity Index (Ri) was calculated using initial absorbance Ainitial (6 s) and final Afinal (540 s), Ri = 1 / (Ai-Af). The Ri of 119 patients (2-17 y / o, M & F) SCA, SCC (Sickle Cell/hemoglobin C), SCD (Sickle Cell/hemoglobin D), Sβ0thal (Sickle Cell/hemoglobin Beta Zero Thalassemia) and 71 blood donors (20-65 y / o, M & F) were measured in our laboratory while the five parameters (TAMMV and PI by TCD, Hct, Hb and RDW) were obtained from medical records. The in vitro addition of hydroxyurea (HU, 50mg /dl, n = 51 patients, and n = 8 healthy donors) in the samples decreased the rouleaux adhesion strength of both donor and patients' blood samples, leading to extraordinarily high Ri values. The correlation between the studied parameters was especially significant for the direct relationships between Ri, TAMMV, and PI.

Уремический синдром вызывает нарушение деформационно-функциональных характеристик эритроцитов у пациентов хронического гемодиализа

End stage of chronic kidney disease (CKD-V) is associated with alterations in erythrocyte structure and functions which are the potential risk factors to interrupt erythrocyte proper activity however the mechanisms of such changes remain understudied. We used laser diffraction approach for investigation of CKD and hemodialysis (HD) effects on deformability of CKD-V patients red blood cells. HD did not affect red blood cell (RBCs) deformability however we showed two-stage alteration in renal patients’ RBCs deformability corresponding to the time of HD treatment or/and susceptibility to uremic syndrome. The first stage was characterized by increase in initial velocity of cell swelling (Vi) and decrease in the time to peak hemolysis rate (Tmax), and indicated the increase in RBCs fragility in patients receiving HD treatment up to 25 months (Group 1). The second stage outlined the decrease in Vi and increase in Tmax, that indicated the increase in rigidity of RBCs in patients receiving HD treatment from 25 months to 250 months (Group 2). In interdialytic periods the increase in concentration of uremic toxins (urea, uric acid) was detected, at the same time there were no changes in sodium and glucose concentrations. The increase in uremic toxins lead to osmolality accrual up to 330 mOsm/kg in patients of Group1 before HD, up to 340mOsm/kg in patients of Group 2 before HD. In summary, HD did not affect the functional parameters of RBCs, however our data clearly indicated the two-stage deformability alteration in renal patients’ RBCs that is triggered by uremic syndrome and potentially may lead to renal anemia and cardio-vascular complications specifically attributed to CKD-V patients.

Cross-sectional focusing of red blood cells in a constricted microfluidic channel.

Constrictions in blood vessels and microfluidic devices can dramatically change the spatial distribution of passing cells or particles and are commonly used in biomedical cell sorting applications. However, the three-dimensional nature of cell focusing in the channel cross-section remains poorly investigated. Here, we explore the cross-sectional distribution of living and rigid red blood cells passing a constricted microfluidic channel by tracking individual cells in multiple layers across the channel depth and across the channel width. While cells are homogeneously distributed in the channel cross-section pre-contraction, we observe a strong geometry-induced focusing towards the four channel faces post-contraction. The magnitude of this cross-sectional focusing effect increases with increasing Reynolds number for both living and rigid red blood cells. We discuss how this non-uniform cell distribution downstream of the contraction results in an apparent double-peaked velocity profile in particle image velocimetry analysis and show that trapping of red blood cells in the recirculation zones of the abrupt construction depends on cell deformability.

Characterization of Bulk Rigidity of Rigid Red Blood Cell Populations in Sickle Cell Patients Using a Parameterization Model of Ektacytometry

Introduction: One of the critical characteristics of sickle-cell disease (SCD) is the increased rigidity of the red blood cell (RBC) membrane. This decreased flexibility of hemoglobin S (HbS)-rich RBCs contributes to reduced lifespan of the cell and is known to be a primary cause of vaso-occlusion, leading to painful crisis episodes and other complications in SCD. Although often overlooked as a factor in disease severity, increased RBC rigidity has been recently shown to reduce the vascular wall adhesion of white blood cells (WBCs) drastically - hinting at a larger role for RBC rigidity in patient well-being beyond instigating vasocclusion. The rigidity and composition of RBCs in SCD patients varies highly, i.e., there can be a large fraction of HbS cells with moderate stiffness or a small fraction of extremely rigid HbS cells. Ektacytometry is a primary technique used for measuring RBC deformability in SCD via laser diffraction viscometry, but is limited in that it renders an average measurement of deformability for the entire RBC population, i.e., across both stiff and healthy RBCs. This average measurement of deformability may underestimate the rigidity of HbS-rich (%S fraction) or sickle RBC population in the blood. Here, we present an approach to estimate rigidity of HbS cells in SCD via a method that implements artificially rigidified RBCs mixed into whole blood at different ratios. Methods: We develop an approach to estimate rigidity of HbS cells in SCD via a method that implements artificially rigidified RBCs. RBCs from healthy (non-SCD) are incubated in solutions with varying concentrations of tert-butyl hydroperoxide to stiffen the cell membrane. These artificially rigidified RBCs are then mixed into whole blood at different ratios/fractions. Four parent concentrations of TBHP are chosen as base RBC rigidities: 1.0, 0.9, 0.75, and 0.5 mM TBHP. After adequate washing, stiffened RBCs are mixed with healthy RBCs in whole blood in increments of 10% rigid RBC fractions up to a total of 100% while holding the hematocrit constant at ~40%. All samples are then measured independently using a LoRRca MaxSis Ektacytometer (Mechatronics Instruments BV, Zwaag, The Netherlands). We then parameterize these ektacytometry deformability measurements to develop a numerical model that predicts the bulk rigidity of the rigid RBC population in SCD blood samples. We measured the EI for fifteen different SCD patient blood samples, details including patient SCD genotype, age, gender, current medical intervention/therapies, %S fraction, and measured maximum bulk EI. Results: With the predicted maximum EI of the rigid RBC population in patient blood samples, we plotted deformability curves using techniques described by Baskurt et al. We utilized a simplified Skreestra-Bronkhorst model for plotting predicted deformability curves implementing critical parameters such as maximum EI, shear where half the maximum EI is achieved, and the range of shear stress in the standard ektacytometry deformability measurement. The predicted bulk SCD whole blood deformability curves are overlaid with the healthy blood curve, experimental bulk SCD whole blood standard measurement, and with the predicted curve of the sickle RBC population for patients 2, 6, and 7 in Figure 1a, 1b, and 1c. These plots show that the precision of the parameterized deformability curves compared to a standard healthy curve and actual measurement of the patient sample. Specifically, in Figure 1a and 1b shows there is large differences in the elongation curve of the sickle population in comparison to the bulk measurements, yielding a maximum EIs that are ~2 times smaller than the bulk measurement. Conversely, in Figure 1c we see there is not much difference in the elongation curve of the sickle population compared to the actual raw standard measurement. Conclusions: Overall, this work presents an innovative method for a more thorough examination of ektacytometry deformability measurements of SCD patients. An understanding of bulk stiffness of the HbS population is crucial as it would allow for more accurate characterization of the impact of RBC rigidity on SCD patient crisis and general wellbeing. More detailed information on the rigidity of the HbS RBC population in SCD has the potential to be utilized to understand phenomena in hemodynamics and connection between RBC rigidity and occurrences of painful crisis in SCD patients. Disclosures No relevant conflicts of interest to declare.

The Effect of Rigid Red Blood Cells on Platelet Adhesion in Blood Flow and Implications in Sickle Cell Disease

Introduction: Sickle cell disease (SCD) occurs due to a mutation in the β-subunit of hemoglobin, causing stiffening of red blood cells (RBCs) and leading to RBC sickling and vaso-occlusive crises (VOC) in SCD patients. While sickled RBCs remain a hallmark of SCD, they are prone to lysis and represent a small fraction of the total RBCs present in patients at a given time. The remaining RBCs maintain a normal, discoid shape and are either healthy or stiff due to polymerization of the hemoglobin β-globin subunit. In healthy blood flow, RBCs form a core in the center of the vessel and the remaining cells, platelets and white blood cells (WBCs), marginate towards the endothelium. However, the increased stiffness of RBCs in SCD disrupts this neat segregation of blood cells to different areas of the blood vessel and can contribute to VOC, the root cause of many acute and chronic complications for SCD patients. Despite the presence of normally shaped, stiffened RBCs in SCD patients, the impact of these RBCs on other cell types in blood flow is currently not well understood. Our laboratory previously demonstrated that the presence of artificially rigidified RBCs leads to an expansion of the RBC core and significantly decreases WBC adhesion to an inflamed endothelium in vitro. Here, we examine the impact of stiffened RBCs on platelet adhesion to a damaged endothelium in vitro by first using a model system with artificially rigidified RBCs and second, utilizing SCD patient blood to further support our model and understand platelet-RBC interactions in SCD patients. Methods: In our model system, we artificially rigidified RBCs taken from healthy donors and reconstituted them into whole blood before perfusing the mixture over an activated, damaged endothelium using a parallel plate flow chamber. We quantified platelet adhesion to the endothelium in comparison to healthy, non-rigidified controls using fluorescent microscopy. To determine if our model findings translated to SCD, we recruited a cohort of hemoglobin SS and SC patients during routine visits and similarly perfused their whole blood over the same damaged endothelium and quantified platelet adhesion. Results and conclusions: The inclusion of artificially rigidified RBCs in otherwise healthy subject blood flow significantly increased platelet adhesion to a damaged endothelium with a maximum increase in platelet adhesion of six-fold over a healthy, non-rigid control in our model system. Both RBC rigidity and the percentage of RBCs that were artificially rigidified had a large impact on the increase in platelet adhesion. SCD platelet adhesion to the damaged endothelium model varied from donor to donor based on variables such as treatment method and disease severity. Overall, this work experimentally elucidates the biophysical impact of stiffened RBCs on platelet adhesion using both an artificial model utilizing healthy blood as well as SCD blood, which can help determine the mechanism of action causing VOC in SCD. Disclosures No relevant conflicts of interest to declare.

Editing a γ-Globin Repressor Binding Site Restores Fetal Hemoglobin Synthesis and Corrects the Phenotype of Sickle Cell Disease Erythrocytes

Sickle cell disease (SCD) is a severe, life-threatening disorder caused by a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) β-chain that causes Hb polymerization with consequent red blood cell (RBC) rigidity, anemia, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) decreases sickling and significantly reduces the clinical severity of SCD. HPFH mutations cluster at several loci in the promoters of the two γ-globin genes, HBG1 and HBG2, and, disrupt binding sites for transcriptional repressors (e.g., BCL11A and LRF), leading to elevated levels of fetal hemoglobin (HbF) representing up to 40% of total hemoglobin tetramers. In addition, SNPs at position -158 of both γ-globin promoters are associated with enhanced γ-globin expression and may identify a putative transcriptional repressor binding site. Here, we used the CRISPR/Cas9 system to mimic HPFH mutations in the HBG promoters by generating insertion and deletions (InDels) leading to disruption of known and putative binding sites for transcriptional repressors via non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) DNA repair mechanisms. Efficient editing of the LRF binding site (≥ 3 γ-globin promoters/cell in >70% of cells) in SCD patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in a robust, virtually pancellular HbF reactivation and a concomitant reduction in βS-globin levels, recapitulating the phenotype of asymptomatic SCD-HPFH patients. Of note, in RBCs derived from edited HSPCs, HbF levels exceeded 40% of total Hb tetramers, suggesting that CRISPR/Cas9 mediated disruption of the LRF binding site is even more potent than naturally occurring HPFH point mutations in reactivating HbF expression. RBCs derived from edited HSPCs displayed HbF levels sufficient to substantially correct the SCD cell phenotype (~80% of non-sickling RBCs). Although larger deletions (in part generated likely by MMEJ) disrupt more efficiently the LRF binding site, shorter InDels mainly produced by NHEJ, the most active repair mechanism in bona fide hematopoietic stem cells (HSCs), were still able to induce a potent γ-globin reactivation and correct the SCD cell phenotype. Interestingly, upon targeting of the -158 site, we observed HBG de-repression, which, however, was mild, indicating that this region contains a sequence that moderately inhibits γ-globin expression in adult cells, consistently with the mild increase in HbF associated with the -158 SNP. To evaluate the efficiency and the safety of this approach in long-term repopulating HSCs, untreated and edited HSPCs were injected into NSG immunodeficient mice. 16 weeks after transplantation, the engraftment of control and HBG-edited cells was comparable, as analyzed in bone marrow, spleen, thymus and blood. Edited HSCs were capable to differentiate into multiple hematopoietic cell types, with no skewing towards any particular lineage. Importantly, xenotransplantation of HSPCs edited using the gRNAs targeting the LRF binding site demonstrated a high editing efficiency in long-term repopulating HSCs (up to 75%) and a modest reduction in the frequency of deletions likely generated by MMEJ. Overall, this study identifies the binding site for the LRF repressor as a novel, potent and safe target for a genome editing-based treatment of SCD. Disclosures El Nemer: Hemanext: Other: Other. Cavazzana:SmartImmune: Other: Founder.

Peripheral blood erythrocytes morphology in ovarian cancer

Aim. To assess the morphology of circulating red blood cells in patients with stage III ovarian cancer.
 Methods. The object of the study was the peripheral blood erythrocytes of primary patients with ovarian cancer (n=25) who had stage III according to International Federation of Gynecology and Obstetrics. Patients were examined in the gynecological department of Ulyanovsk Regional Clinical Oncology Center. The control group included 25 somatically healthy women. Morphological studies were performed using light microscopy. The number of red blood cells with an unchanged and altered shape was expressed as a percentage. By the method of atomic force microscopy, the topology and rigidity of red blood cells was studied.
 Results. A statistically significant decrease in the number of circulating blood erythrocytes was found in patients with ovarian cancer compared to somatically healthy women. At the same time, the number of discocytes is markedly reduced while the number of morphologically altered forms: echinocytes, stomatocytes, spherocytes and erythrocyte rigidity are increased.
 Conclusion. With the appearance of altered forms of red blood cells and increase of the transformation index and erythrocyte rigidity in patients with stage III ovarian cancer, total number of red blood cells decreases in circulating blood compared to somatically healthy women.

PB2301 ASSESSMENT OF SICKLE PAIN MANAGEMENT IN THE WHITTINGTON HOSPITAL EMERGENCY DEPARTMENT

Background:Sickle cell anaemia (SCA) is an autosomal recessive disorder caused by point mutation of the β‐globin gene, resulting in abnormal forms of hemoglobin that cause increased red blood cell rigidity and hemolysis. It is one of the most common hereditary blood conditions, affecting over 14,000 adults in the UK.One of the manifestations of SCA is vaso‐occlusive crises. These typically cause severe pain that may require emergency department (ED) attendance for pain management, typically with opioids. Those patients in whom pain relief is not well‐controlled are at risk of further complications including acute chest syndrome.The UK National Institute of Clinical Excellence (NICE) published a quality standard in 2014 stating that patients presenting to hospital with an acute painful sickle cell episode should have a pain assessment, a clinical assessment and appropriate analgesia within 30 minutes of presentation.Aims:This study was performed to assess the Whittington Hospital's compliance to national recommendations and try to establish what aspects of care in ED contribute to any delays in management noted.Methods:If a Whittington SCA patient attends ED, an automated email is generated that notifies the haematology team of this. This system was used to identify acute sickle cell presentations to ED. Criteria for inclusion in the study was Whittington SCA patients that presented to ED with acute painful sickle cell crises between August 2017 to January 2018. Patients who received analgesia in the ambulance and patients with no documentation available were excluded. The time of presentation, analgesia prescription and administration for each attendance were noted from ED documentation.Results:A total of 104 ED SCA attendances were included. 41% of patients presenting with an acute painful sickle cell crisis received analgesia during their first 30 minutes in ED. The average wait for analgesia was 47 minutes, with 75% of SCA patients receiving analgesia within 1 hour of arrival.The time taken to triage SCA patients was on average 8 minutes (range 0‐29 minutes). Time from arrival to prescription of pain relief was much more variable with an average wait 40 minutes (range 10 minutes to 2 hours 22 minutes). Patients who frequently attended Whittington ED (defined as 7 or more attendances within the 6 month period studied) had a shorter average wait for analgesia. Analgesia was given 36 minutes after arrival on average for frequent attenders, whereas patients presenting 6 or fewer times had an average wait of 53 minutes.Summary/Conclusion:We are not currently meeting our audit standard for provision of analgesia in the emergency department, and performance appears to have worsened progressively since our earliest available date from 2012 (although methodological differences may have contributed to this). In 2015 49% of patients received analgesia within 30 minutes compared to 41& 2017/18. Most of the delay appears to be due to the time taken for medication to be prescribed, although the time taken to triage the patient and administer the medication was also not insignificant, and often amounted to greater than 30 minutes. It is unclear what is contributing to this delay, although it appears that performance is improved when the patient is a repeat attender and therefore known to the department. Educations sessions with the ED department may improve management of SCA painful crises in ED.

Studying the rigidity of red blood cells induced by Plasmodium falciparum infection

We study the effect of different chemical moieties on the rigidity of red blood cells (RBCs) induced by Plasmodium falciparum infection, and the bystander effect previously found. The infected cells are obtained from a culture of parasite-infected RBCs grown in the laboratory. The rigidity of RBCs is measured by looking at the Brownian fluctuations of individual cells in an optical-tweezers trap. The results point towards increased intracellular cyclic adenosine monophosphate (cAMP) levels as being responsible for the increase in rigidity.

The relationship between the rheological behavior of RBCs and angiogenesis in the morbidly obese.

The aim of this study was to investigate the relationship between red blood cell (RBC) aggregation and deformability and angiogenesis parameters in obese patients. We studied 35 obese subjects and 20 non-obese people as a control group. Angiogenesis was detected using Bio-Plex Pro Human Angiogenesis Multiplex Assays. The RBC aggregation and deformability of the red blood cell aggregation were performed by the Laser-assisted Optical Rotational Cell Analyser - LORCA. The aggregation index and the syllectogram's amplitude were significantly higher in the obese patients, whereas the aggregation half-time (t1/2) was lower compared with the control group. The deformability of RBC expressed as EI was significantly lower in the obese group than it was in the control group. All angiogenesis parameters were higher in obese individuals than they were in the control group. Significant differences were observed in angiopoietin 2 (p = 0.048), folistin (p = 0.0017), G-CSF (p = 0.042), HGF (p = 0.016), and PECAM-1 (p = 0.014). The VEGF tended to be higher in the obese patients than in the control group (p = 0.09); nevertheless, the concentration of PDGF-BB was similar in both groups. EI at shear stresses of 18.49 Pa and 30.2 Pa was strongly correlated with all angiogenesis parameters. No correlations were found between the studied RBC aggregation indices and angiogenesis parameters. Multivariate analyses indicated that only HGF was an independent predictor of RBC deformability at 18.49 Pa (β-0.83, P < 0.000005) and at 30.2 Pa (β-0.83, P < 0.00005). The study found that there are relationships between enhanced RBC rigidity and angiogenesis status in obese subjects. Because this correlation between angiogenesis and RBC deformability is presented for the first time, the physiological importance of the relationship requires further research.

Towards Mechanical Clinical Markers in Sickle Cell Disease: Dynamics of Red Blood Cells in Low Shear Flow

Several regimes of motion are evident in red blood cells (RBCs) under shear flow and are known to be controlled by the shear flow rate and by the deformability of the cell. Above a certain shear flow rate, healthy RBCs present a fluidized regime analogous to that of a droplet: the membrane adopts a tank-treading motion, i.e. rotates around the centre of mass of the cell, and its orientation oscillates around a mean value (Fig. 1). This motion is not observed in rigid RBCs which keep the same regime of motion as with low shear flow rate. The transition from one motion to the other depends on several intrinsic parameters including membrane viscosity, elasticity and cytoplasmic viscosity.Sickle cell disease (SCD) is a hereditary hemolytic anemia due to the presence of mutant hemoglobin, Hb S, which tends to polymerize in RBCs, reducing its deformability and resulting ultimately in RBC sickling. We postulate that SCD may affect RBCs capacity to adopt the tank-treading motion.Thanks to microfluidic chips coupled with high speed video, we studied the fraction of RBCs displaying a tank-treading motion (% of TT) at moderate shear stresses (0.4-0.6 Pa), in homozygous SCD patients (n=30) and in normal subjects (n=12). At the time of sampling, SCD patients were in steady state (no vaso-occlusive crisis or other acute complications) and not transfused for at least 3 months. Around 3 microL of total blood were necessary for each measurement. RBCs were studied under atmospheric conditions and exhibited the normal discoid shape.We showed first that the % of TT is significantly different in SCD patients compared to controls, with no overlap between the values of each groups (respectively 70.4±12.9% versus 98.6±0.9%). Second, measurements performed on different cells fractions isolated by cell density or in the presence of different NaCl concentrations, showed that the % of TT cells is sensitive to cell density and hydration status, both in normal subjects and in SCD patients. Finally, we evaluate the % of TT in RBC samples drawn just before or during 10 episodes of vaso-occlusive crisis from 8 different patients. The definition of a vaso-occlusive crisis, for this specific study, was set as an episode with pain in at least 2 different locations requiring hospitalization at least for 24h. The results revealed that, for a given patient, the % of TT cells varies significantly for 12h before as well as during vaso-occlusive crises with kinetics comparable from one patient to another.In conclusion, we described here a new inexpensive biological test able to discriminate healthy RBCs from sickle RBCs, with preliminary results suggesting potential interest for monitoring the clinical status of SCD patients. Further analysis will be necessary to determine if this parameter is suitable for predicting the occurrence of vaso-occlusive crises early enough to allow preventive actions.Fig1: Schematic view of the regime of motion corresponding to tank-treading. Flow direction is from left to right and RBCs are observed from the direction of the shear gradient. The black dot on the RBCs displays the motion of a membrane element on the RBC surface. The axis of symmetry remains in the shear plane, the body of the RBC oscillates slightly as the membrane rotates around it DisclosuresThuret:Addmedica: Research Funding; bluebird bio: Research Funding; Novartis: Research Funding.

Assessment of Sickle Cell Disease Pain Management in the Whittington Hospital Emergency Department

Background and aimsSickle cell anaemia (SCA) is an autosomal recessive disorder caused by point mutation of the β-globin gene, resulting in abnormal forms of hemoglobin that cause increased red blood cell rigidity and hemolysis. It is one of the most common hereditary blood conditions, affecting over 14,000 adults in the UK (Dormandy el al, 2017).One of the manifestations of SCA is vaso-occlusive crises. These typically cause severe pain that may require emergency department (ED) attendance for pain management, typically with opioids. Pain relief should be given quickly and response to this assessed on a regular basis to ensure pain management is optimized. Those patients in whom pain relief is not well-controlled are at risk of further complications including acute chest syndrome.The UK National Institute of Clinical Excellence (NICE) published a quality standard in 2014 stating that patients presenting to hospital with an acute painful sickle cell episode should have a pain assessment, a clinical assessment and appropriate analgesia within 30 minutes of presentation (NICE, 2014).This study was performed to assess the Whittington Hospital's compliance to national recommendations and to establish which aspects of care in ED contributed to delays in management.MethodsIf a Whittington SCA patient attends ED, an automated email is generated that notifies the haematology team of the attendance. This system was used to identify acute sickle cell presentations to ED. Criteria for inclusion in the study was Whittington SCA patients that presented to ED with acute painful sickle cell crises between August 2017 to January 2018. Patients who received analgesia in the ambulance and patients with no documentation available were excluded. The time of presentation, analgesia prescription and administration for each attendance were noted from ED documentation.ResultsA total of 104 ED SCA attendances were included. 41% of patients presenting with an acute painful sickle cell crisis received analgesia during their first 30 minutes in ED. The average wait for analgesia was 47 minutes, with 75% of SCA patients receiving analgesia within 1 hour of arrival.The time taken to triage SCA patients was on average 8 minutes (range 0 to 29 minutes). Time from arrival to prescription of pain relief was much more variable with an average wait 40 minutes (range 10 minutes to 2 hours 22 minutes). Time from prescription to administration also varied, with 56% administered within 10 minutes of prescription and 87% within 30 minutesPatients who frequently attended Whittington ED (defined as 7 or more attendances within the 6 month period studied) had a shorter average wait for analgesia. Analgesia was given 36 minutes after arrival on average for frequent attenders, whereas patients presenting 6 or fewer times had an average wait of 53 minutes.ConclusionsWe are not currently meeting our audit standard for provision of analgesia in the emergency department, and performance appears to have worsened progressively since our earliest available date from 2012 (although methodological differences may have contributed to this). In 2015 49% of patients received analgesia within 30 minutes compared to 41% in 2017/18. Most of the delay appears to be due to the time taken for medication to be prescribed, although the time taken to triage the patient and administer the medication was also not insignificant and often amounted to greater than 30 minutes. It is unclear what is contributing to this delay, although it appears that performance is improved when the patient is a repeat attender and therefore known to the department. Educations sessions with the ED department and availability of a SCA specialist nurse may improve management of SCA painful crises in ED. DisclosuresShah:Novartis: Honoraria, Speakers Bureau; Sobi/Apotex: Honoraria; Celgene Corp: Other: Steering committee; Roche: Other: Advisory board meeting.

Numerical simulation on effect of red blood cell motion on flow behavior of liposome-encapsulated hemoglobin

We simulated flows in microvascular vessels containing deformable red blood cells (RBCs) and liposome-encapsulated hemoglobin (LEH) particles. Blood flow was computed by the lattice Boltzmann method (LBM), and fluid–membrane interaction between the flow field and RBCs was incorporated using the immersed boundary method (IBM). The simulation results indicated that the LEH dispersion in a direction perpendicular to the main flow was largely affected by the presence of RBCs. In particular, LEH dispersion was high in the presence of rigid RBCs with low deformability. These results could provide essential information for designing LEH-mediated efficient oxygen supply to tissues.

Vascular-targeted particle binding efficacy in the presence of rigid red blood cells: Implications for performance in diseased blood.

The field of drug delivery has taken an interest in combating numerous blood and heart diseases via the use of injectable vascular-targeted carriers (VTCs). However, VTC technology has encountered limited efficacy due to a variety of challenges associated with the immense complexity of the in vivo blood flow environment, including the hemodynamic interactions of blood cells, which impact their margination and adhesion to the vascular wall. Red blood cell (RBC) physiology, i.e., size, shape, and deformability, drive cellular distribution in blood flow and has been shown to impact VTC margination to the vessel wall significantly. The RBC shape and deformability are known to be altered in certain human diseases, yet little experimental work has been conducted towards understanding the effect of these alterations, specifically RBC rigidity, on VTC dynamics in physiological blood flow. In this work, we investigate the impact of RBCs of varying stiffnesses on the adhesion efficacy of particles of various sizes, moduli, and shapes onto an inflamed endothelial layer in a human vasculature-inspired, in vitro blood flow model. The blood rigid RBC compositions and degrees of RBC stiffness evaluated are analogous to conditions in diseases such as sickle cell disease. We find that particles of different sizes, moduli, and shapes yield drastically different adhesion patterns in blood flow in the presence of rigid RBCs when compared to 100% healthy RBCs. Specifically, up to 50% reduction in the localization and adhesion of non-deformable 2 μm particles to the vessel wall was observed in the presence of rigid RBCs. Interestingly, deformable 2 μm particles showed enhanced vessel wall localization and adhesion, by up to 85%, depending on the rigidity of RBCs evaluated. Ultimately, this work experimentally clarifies the importance of considering RBC rigidity in the intelligent design of particle therapeutics and highlights possible implications for a wide range of diseases relating to RBC deformability.

Blood rheology during normal pregnancy.

Recent studies have shown increased RBC aggregation and no difference in plasma viscosity in the presence of markedly lower hematocrit in women at term compared to non-pregnant women. Little is known about the outcome of blood rheological parameters and red blood cell (RBC) deformability particularly in the course of normal pregnancy. During a 36 months interval 1.913 blood samples were randomly collected from a total of 945 pregnant women in the course of their pregnancy (n = 1.259) and during puerperium (upto 1 week; n = 654). Next to the blood count, hemorheological parameters including red blood cell (RBC) -aggregation (stasis E0; low shear E1), -deformability (low, moderate and high shear conditions) and plasma viscosity (pv) were assessed. Plasma viscosity (pv) was examined using KSPV 1 Fresenius, RBC aggregation (stasis: E0 and low shear: E1) using MA1-Aggregometer; Myrenne and RBC deformability (def) was determined by Rheodyn SSD Diffractometer, Myrenne, Roetgen, Germany were tested. In some of these women laboratory results prior to pregnancy (n = 145) were available which were compared with those during pregnancy. Mean maternal pv remained unchanged within each trimester and compared to the values before pregnancy and during early puerperium (Range of means: 1.18-1.20 mPa S). In contrast, RBC agg (E0 and E1) was markedly higher in the 2nd (21.8 ± 7.0 and 28.9 ± 9.4; p < 0.001) and 3rd trimester (18.74 ± 8.4 and 28.2 ± 9.4; p < 0.01) compared to the values before pregnancy (16.4 ± 6.4 and 20 ± 7.5) and during 1st trimester (17.49 ± 6.5 and 22.4 ± 7.4). There was a stat. significant temporary reduction in RBC def. under all shear rate conditions during 2nd trimester compared to the values before pregnancy which remained significantly lower during 3rd trimester only under high shear rates.An increase RBC agg was stat. significantly inversely correlated with reduced RBC def being most pronounced under low shear rate conditions. While RBC rigidity was stat. significantly correlated with higher hematocrit values there was only a weak correlation between RBC agg and haematocrit (E0: r = -0.084; p = 0.03; E1: r = -0.06; p = 0.1). Pv was not correlated with haematocrit or RBC def but stat. significantly correlated with RBC agg. Blood rheological changes manifest during 1st trimester, and fairly remain unchanged during 2nd trimester until term. Physiologic hemodilution and increasing hypercoagulability is accompanied by high RBC -aggregation and - rigidity during 2nd trimester while plasma viscosity remains nearly unaffected throughout normal pregnancy.

Presence of Rigid Red Blood Cells in Blood Flow Interferes with the Vascular Wall Adhesion of Leukocytes.

The symptoms of many blood diseases can often be attributed to irregularities in cellular dynamics produced by abnormalities in blood cells, particularly red blood cells (RBCs). Contingent on the disease and its severity, RBCs can be afflicted with increased membrane rigidity as seen in malaria and sickle cell disease. Despite this understanding, little experimental work has been conducted toward understanding the effect of RBC rigidity on cellular dynamics in physiologic blood flow. Though many have computationally modeled complex blood flow to postulate how RBC rigidity may disrupt normal hemodynamics, to date, there lacks a clear understanding of how rigid RBCs affect the blood cell segregation behavior in blood flow, known as margination, and the resulting change in the adhesion of white blood cells (WBCs). In this work, we utilized an in vitro blood flow model to examine how different RBC rigidities and volume fractions of rigid RBCs impact cell margination and the downstream effect on white blood cell (WBC) adhesion in blood flow. Healthy RBC membranes were rigidified and reconstituted into whole blood and then perfused over activated endothelial cells under physiologically relevant shear conditions. Rigid RBCs were shown to reduce WBC adhesion by up to 80%, contingent on the RBC rigidity and the fraction of treated RBCs present in blood flow. Furthermore, the RBC core was found to be slightly expanded with the presence of rigid RBCs, by up to ∼30% in size fully composed of rigid RBCs. Overall, the obtained results demonstrate an impact of RBC rigidity on cellular dynamics and WBC adhesion, which possibly contributes to the pathological understanding of diseases characterized by significant RBC rigidity.