Red Blood Cell Fraction Research Articles

Dielectrophoretic microfluidic device for separation of red blood cells and platelets: a model-based study

The paper proposes a dielectrophoresis microfluidic chip for particle separation, which uses dielectric properties to perform size-based fractionation of red blood cells and platelets. Based on the control variables, the distribution of the electric field in the chip and the trajectory of the particles in the microfluidic channel are calculated using COMSOL Multiphysics under different electrode shapes, voltages and chip exit structures. Both red blood cells and platelets respond to negative dielectrophoresis at an alternating current signal with a frequency of 100 kHz. The larger red blood cells are subjected to a stronger dielectrophoretic force than the platelets and are biased toward the right outlet, and the platelets flow out from the left outlet under the combined action of fluid force and dielectrophoretic force to achieve the purpose of separation. On this basis, through quantitative comparison and analysis, a more optimized microfluidic chip capable of effectively separating particles is finally selected.

In vitro investigations of red blood cell phase separation in a complex microchannel network

Microvascular networks feature a complex topology with multiple bifurcating vessels. Nonuniform partitioning (phase separation) of red blood cells (RBCs) occurs at diverging bifurcations, leading to a heterogeneous RBC distribution that ultimately affects the oxygen delivery to living tissues. Our understanding of the mechanisms governing RBC heterogeneity is still limited, especially in large networks where the RBC dynamics can be nonintuitive. In this study, our quantitative data for phase separation were obtained in a complex in vitro network with symmetric bifurcations and 176 microchannels. Our experiments showed that the hematocrit is heterogeneously distributed and confirmed the classical result that the branch with a higher blood fraction received an even higher RBC fraction (classical partitioning). An inversion of this classical phase separation (reverse partitioning) was observed in the case of a skewed hematocrit profile in the parent vessels of bifurcations. In agreement with a recent computational study [P. Balogh and P. Bagchi, Phys. Fluids 30,051902 (2018)], a correlation between the RBC reverse partitioning and the skewness of the hematocrit profile due to sequential converging and diverging bifurcations was reported. A flow threshold below which no RBCs enter a branch was identified. These results highlight the importance of considering the RBC flow history and the local RBC distribution to correctly describe the RBC phase separation in complex networks.

Fibrinogen-Mediated Platelet Microaggregate Formation in Stored Whole Blood

Introduction: Whole blood is regaining popularity in resuscitation after hemorrhage. The platelet storage lesion that has been observed in apheresis platelets also exists in whole blood, where platelet count and aggregation response decline rapidly after collection and during storage in the blood bank. Platelet microaggregates form in unfiltered whole blood, resulting in relatively large but unstable thrombus formation in a flow model over collagen. We hypothesized that the presence of fibrinogen activates platelets within the whole blood unit and predisposes them toward premature aggregate formation. Methods: Fresh whole blood (WB) was collected in citrate tubes (4.3%, 10.9 mM) from healthy donors (n=4) according to an institutionally approved standard operating procedure. Blood was centrifuged at 200-g to collect the platelet-rich plasma (PRP). The remaining red blood cell (RBC) fraction was centrifuged at 2000-g to pellet RBCs which were washed with HEPES-buffered saline and repelleted; platelet-poor plasma (PPP) was saved. The PRP fraction was centrifuged with 1 µM prostacyclin at 700-g, and the platelet pellet was washed with Tyrode's buffer, repelleted, and resuspended in either fibrinogen-deficient plasma (Fg-DP) or PPP. Platelets were combined with washed RBCs to generate a reassembled whole blood sample containing 150,000 platelets/µl and a hematocrit of 40%. Samples were added to minibags and stored at 4 °C for 14 days. On days of testing (0, 3, 7, 14), 1 ml aliquots were stained with 1 µM calcein-AM for 30 min at 37 °C and run on the BioFlux 1000 microfluidics platform with arterial flow rates generating a shear rate of 980 s-1 over a collagen surface. Fluorescence microscopy images were captured and quantified over 10 min to observe platelet adhesion to the surface. Complete blood count (CBC) was measured on each aliquot. In a follow-up study, unmanipulated, stored whole blood was passed through a standard transfusion filter (200 µm pores) to determine the effects of removing microaggregates prior to microfluidics flow testing at each time point. Results: CBC results are in Figure 1, illustrating the decline of platelet count over storage as previously observed. Figure 2 shows representative images captured at the end of the 10 min flow period. In samples containing normal plasma, thrombus formation is impaired by day 3 of storage with aggregate structures appearing immediately in the microscopy images. These aggregates attach and release periodically over the course of flow, an effect that persisted through day 14. In samples reconstructed with Fg-DP, no microaggregates were observed during storage, although the density and size of thrombi diminished over time. In filtered whole blood samples, thrombus formation on days 3 and 7 more closely resembled the Day 0 fresh samples, but aggregates appeared on day 14 despite filtration. Discussion: WB contains all of the elements required for resuscitation after hemorrhage, however the platelet storage lesion may affect its function. The interaction of platelets and fibrinogen in a relatively static environment results in activation and aggregation of platelets. In apheresis units, studies have shown that using a platelet additive solution to dilute fibrinogen results in better platelet performance for longer periods of storage. Further study is required to determine the clinical significance of aggregate formation and whether mitigation strategies should be explored. Disclosures No relevant conflicts of interest to declare.

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.

Type 1 IFN signaling critically regulates influenza-induced alloimmunization to transfused KEL RBCs in a murine model.

Only a fraction of red blood cell (RBC) transfusion recipients form alloantibodies, and variables determining responsiveness or nonresponsiveness are poorly understood. We and others have previously shown in animal models that pretreatment with toll-like receptor agonists that mimic different types of infections impacts the magnitude or frequency of RBC alloantibody responses. We hypothesized that influenza infection, coexistent with transfusion, would impact responses to transfused RBCs in a manner dependent on Type 1(α/β) interferon (IFN) signaling and tested this in a murine model. Wild-type mice or mice lacking the ability to respond to Type 1 IFN were infected with influenza prior to the transfusion of transgenic murine RBCs (K1) expressing the human KEL glycoprotein or the triple fusion HOD protein. Alloantibody responses were measured longitudinally after transfusion by flow cytometric crossmatch, and posttransfusion RBC recovery and survival was evaluated. Influenza-infected mice transfused with K1 RBCs developed robust anti-KEL alloantibodies, whereas animals transfused in the absence of infection remained nonresponders; influenza-associated RBC alloimmunization was also observed after transfusion of HOD RBCs. Recipient Type 1 IFN production was critical to the mechanism of action of influenza-induced RBC alloimmunization, with alloimmunization being significantly decreased in mice unable to sense Type 1 IFN (through antibody blockade or genetic approaches). These and other data suggest that Type 1 IFN responses to toll-like receptor agonists or infections regulate RBC alloantibody responses. Studies investigating whether such a correlation exists in humans may be informative.

Blood Circulating Exosomes Contain Distinguishable Fractions of Free and Cell-Surface-Associated Vesicles

Considering exosomes as intercellular transporters, inevitably interacting with the plasma membrane and the large available surface of blood cells, we wonder if a fraction of circulating exosomes is associated with the surface of blood cells. The aim of this study was to develop an efficient protocol for isolating exosomes associated with the surface of blood cells and to further investigate the characteristics of this fraction in a healthy state and during the development of breast cancer, as well as its possible implication for use in diagnostic applications. Blood samples were collected from Healthy Females (HFs) and breast cancer patients (BCPs). Exosomes extracted from blood plasma and eluted from the surface of blood cells were isolated by ultrafiltration with subsequent ultracentrifugation. Transmission Electron Microscopy (TEM), along with immunogold labeling, demonstrated the presence of exosomes among membrane-wrapped extracellular vesicles (EVs) isolated from both plasma and blood cell eluates. TEM, nanoparticle tracking analysis, and NanoOrange protein quantitation data showed that cell-associated exosomes constituted no less than 2/3 of total blood exosome number. Exosomes, ranging from 50-70 nm in size, prevailed in the blood of breast cancer patients, whereas smaller exosomes (30-50 nm) were mostly observed in the blood of healthy women. Analysis of specific proteins and RNAs in exosomes circulating in blood demonstrated the significant differences in the packing density of the polymers in exosomes of HFs and BCPs. Preliminary data indicated that detection of cancer-specific miRNA (miR-103, miR-191, miR-195) in exosomes associated with the fraction of red blood cells allowed to discriminate HFs and BCPs more precisely compared to cell-free exosomes circulating in plasma. Our data provide the basis for using blood cell-associated exosomes for diagnostic applications.

RETRACTED: A new idea of Atangana-Baleanu time fractional derivatives to blood flow with magnetics particles in a circular cylinder: Two phase flow model

This study aims to examine the magnetohydrodynamic (MHD) effect on the blood flow of a Casson fluid in a vertical cylinder. Blood is taken as a non-Newtonian viscous fluid due to the viscosity which depends on the volume fraction of red blood cells. The effect of magnetic field on blood and particles velocities with temperature distribution has been studied. Using the definition of Atangana-Baleanu fractional derivatives, the classical model which governs the flow has been generalized and the exact solution of the problem has been obtained by applying the joint transformation of Laplace and Hankel. Effects of interesting embedded parameters on both velocities (blood velocity and particles velocity) have been examined and discussed graphically. Results showed that an increase in blood velocity or particle velocity is caused due to increase in Gr and λ, however, a reverse behavior is noticed for higher values of M. Rate of heat transfer at the plate surface is measured using Nusselt number relation. The results are computed and presented in a tabular form.

Effects of shear-dependent viscosity and hematocrit on blood flow

In this paper, we have proposed a new non-linear constitutive model for blood with a viscosity which depends on the shear rate and the volume fraction of red blood cells or RBCs (hematocrit). The viscosity correlation is obtained from the experimental data reported in the literature. The governing equations for flow between two flat plates are made dimensionless and solved numerically. A parametric study is performed to study the effect of the four non-dimensional numbers K1 (a relative measure of the pressure gradient to viscous forces), K2 (a relative measure of gravitational force to pressure gradient in the direction of the gravity), N (dimensionless average hematocrit of blood), and ϕm (maximum packing fraction of RBCs). The results show good qualitative agreement with other studies.

Genetic deletion of the DNA damage repair protein, ATM kinase, is not sufficient to induce vascular dysfunction in young mice

Ataxia telangiectasia mutated (ATM) kinase orchestrates the cellular response to double stranded DNA breaks, phosphorylating and activating downstream targets that lead to cell cycle arrest or apoptosis, as well as by recruiting the machinery necessary for DNA repair. In the vasculature, ATM may play a critical role in maintaining genomic stability, thereby preserving vascular function, as aberrant DNA repair has been implicated in vascular dysfunction. Here, we evaluated several measures of vascular function in ATM knockout (ATM −/−) and wildtype (ATM +/+) mice (4–6 mo, n=7–9/group). Aortic pulse wave velocity, a measure of large artery stiffness, was similar between groups (P>0.05). Furthermore, using an automated capture and analysis system we assessed markers of microvascular perfusion and glycocalyx properties. Perfused microvascular density, a measure of the number of perfused microvessels in a given area, and red blood cell (RBC) fraction, a marker of the heterogeneity of RBCs between microvessel segments, were not significantly different between groups (P>0.05). Likewise, perfused boundary region, a measure of endothelial glycocalyx barrier function, was not significantly different between ATM −/− and ATM +/+ controls (P>0.05). Furthermore, we examined endothelium‐dependent dilation and nitric oxide (NO) bioavailability in mesenteric arteries by measuring vasodilation to acetylcholine (ACh) in the absence and presence of the nitric oxide synthase inhibitor, L‐NAME. Maximal vasodilation to ACh did not differ between groups (P>0.05). Likewise, the contribution of NO to maximal vasodilation was similar between ATM −/− and ATM +/+ mice (P>0.05). Next, we assessed smooth muscle responsiveness to NO by measuring vasodilation to the NO donor, sodium nitroprusside (SNP). Maximal vasodilation to SNP did not differ between groups (P>0.05). Lastly, we evaluated the passive mechanical properties of isolated mesenteric arteries and found no differences in vessel lumen diameter in response to increasing pressure between ATM −/− and ATM +/+ mice (P>0.05). In conclusion, these findings suggest that removal of a major kinase involved with double stranded DNA damage signaling does not affect vascular function in young mice. This could be due to several reasons. First, young mice may have not accumulated sufficient DNA damage and mutagenesis to induce dysfunction or second, signaling downstream from DNA damage may drive vascular dysfunction. Thus, future studies are warranted that examine the role of ATM kinase in vascular function in response to conditions that augment DNA damage.Support or Funding InformationFunding Information: Funded in part by awards from the NIH, R01 AG048366, R01 AG050238, K02 AG045339 and K99 AT010017, and by US Department of Veterans Affairs 1I01BX002151.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Changes in Erythrocyte Morphology at Initiation of Cardiopulmonary Bypass Without Blood Transfusion Were Not Associated With Less Deformability During Cardiac Surgery

During cardiac surgery, circulating red blood cells (RBCs) are at risk of exposure to environmental factors during extracorporeal circulation and transfusion of stored RBCs. For this study, the authors observed morphological differences, deformability, density distribution, and erythrocyte indices of RBCs during cardiac surgery with cardiopulmonary bypass (CPB). Prospective study. Tertiary care center affiliated with a university hospital. Adults who underwent elective cardiac surgery requiring CPB. Blood samples were obtained from 13 patients before incision (baseline), at initiation of CPB, after separation from CPB, and at completion of surgery. The morphological index (MI) in RBCs using light microscopy and the maximum deformability index (DImax) using an ektacytometer were evaluated. In addition, the fractionation of RBCs and erythrocyte indices were measured. The MI at initiation of CPB was significantly higher without blood transfusion compared with baseline, although the DImax did not significantly decrease simultaneously. The DImax after separation from CPB and at completion of surgery were significantly lower than that at baseline. This lowered DImax was accompanied by a significantly reduced mean corpuscular volume and elevated mean corpuscular hemoglobin concentration compared with baseline. Dense RBC subpopulations increased after initiating CPB. The MI after separation from CPB and at completion of surgery partially recovered. Administered stored RBCs showed a high MI and the lowest DImax. Morphological changes at initiation of CPB are considered potentially reversible transformations without loss of the membrane surface area and do not have a significant effect on the DImax. A decrease in deformability likely is due to transfusion of stored RBCs.

Numerical simulation of two-phase non-Newtonian blood flow with fluid-structure interaction in aortic dissection

The behavior of blood cells and vessel compliance significantly influence hemodynamic parameters, which are closely related to the development of aortic dissection. Here the two-phase non-Newtonian model and the fluid-structure interaction (FSI) method are coupled to simulate blood flow in a patient-specific dissected aorta. Moreover, three-element Windkessel model is applied to reproduce physiological pressure waves. Important hemodynamic indicators, such as the spatial distribution of red blood cells (RBCs) and vessel wall displacement, which greatly influence the hemodynamic characteristics are analyzed. Results show that the proximal false lumen near the entry tear appears to be a vortex zone with a relatively lower volume fraction of RBCs, a low time-averaged wall shear stress (TAWSS) and a high oscillatory shear index (OSI), providing a suitable physical environment for the formation of atherosclerosis. The highest TAWSS is located in the narrow area of the distal true lumen which might cause further dilation. TAWSS distributions in the FSI model and the rigid wall model show similar trend, while there is a significant difference for the OSI distributions. We suggest that an integrated model is essential to simulate blood flow in a more realistic physiological environment with the ultimate aim of guiding clinical treatment.

Does endurance training improve red blood cell aging and hemorheology in moderate-trained healthy individuals?

ObjectiveTo examine the impact of a 6-week endurance training on red blood cell (RBC) aging and deformability of healthy participants to detect possible improved hemorheological and performance-related adaptations. MethodsA total of 31 participants (17 females and 14 males) performed a 6-week moderate training protocol (three 1-h running sessions per week at 70% of maximal heart rate). Blood was sampled before and after the training. RBCs from each participant were fractioned according to density and age into 4 RBC subfractions. Subfractions were examined for changes of RBC properties, including aging distribution, RBC deformability, RBC microparticles, and phosphatidylserine concentrations. RBC and plasma nitrite levels were measured as indicators of nitric oxide metabolism. ResultsAerobic performance, peak oxygen consumption, ventilatory thresholds, velocity at the aerobic–anaerobic threshold, and lactate at exhaustion improved after training. The relative amount of both young RBCs and old RBCs increased, and the amount of the main RBC fraction decreased. Phosphatidylserine externalization and RBC-derived microparticles decreased. Overall deformability expressed as shear stress required to achieve half-maximum deformation to theoretical maximal elongation index at infinite shear stress improved in unfractioned RBCs (p < 0.001). Nitrite decreased in total (p = 0.001), young (p < 0.001), main (p < 0.001), and old (p = 0.020) aged RBCs and in plasma (p = 0.002), but not in very old RBCs. ConclusionThese results indicate that non-endurance-trained healthy participants benefit from a regular moderate running training program because performance-related parameters improve and a younger RBC population with improved RBC properties is induced, which might support oxygen supply in the microcirculation.

Small Red Blood Cell Fraction on the UF-1000i Urine Analyzer as a Screening Tool to Detect Dysmorphic Red Blood Cells for Diagnosing Glomerulonephritis.

BackgroundDysmorphic red blood cells (dRBCs) are first-line biomarkers for detecting glomerulonephritis (GN) in patients with hematuria. The UF-1000i system (Sysmex, Kobe, Japan), based on flow cytometry, provides small red blood cell (RBC) values (UF-1000i [UF]-%sRBCs). We evaluated the clinical application of UF-%sRBCs for detecting %dRBCs and GN.MethodsUrine samples of 103 patients (47 with GN; 56 without GN [NGN]) were analyzed using UF-1000i urinalysis, phase-contrast microscopy (PCM), and urine chemistry. Serum creatinine (mg/dL), serum albumin (g/dL), serum protein (mg/dL), urine protein (mg/dL), and urea nitrogen (mg/dL) levels were measured using an automated chemical analyzer. To determine the cut-off level of predicting GN, ROC curve was analyzed.ResultsUF-%sRBCs, %dRBCs, urine protein, serum creatinine, and estimated-glomerular filtration rate differed between the GN and NGN groups, with the greatest differences detected for UF-%sRBCs and %dRBCs (P<0.0001). In ROC curve analysis, urine protein had the highest area under the curve (0.828), followed by %dRBCs (0.771) and UF-%sRBCs (0.745). To screen for GN, the best cut-off values of UF-%sRBCs and %dRBCs were >40.5% and >6.7%, respectively. %dRBCs (P=0.0001) and UF-%sRBCs (P=0.0006) differed between the GN and NGN groups in patients with isolated hematuria but without proteinuria.ConclusionsUF-%sRBCs had similar diagnostic power to %dRBCs determined by PCM for identifying patients with GN. UF-%sRBCs may be more useful for diagnosing GN in patients with isolated hematuria. Predicting %dRBCs using UF-1000i will provide information on possible GN in patients presenting with asymptomatic hematuria.

Small Red Blood Cell Fraction on the UF-1000i Urine Analyzer as a Screening Tool to Detect Dysmorphic Red Blood Cells for Diagnosing Glomerulonephritis

Small Red Blood Cell Fraction on the UF-1000i Urine Analyzer as a Screening Tool to Detect Dysmorphic Red Blood Cells for Diagnosing Glomerulonephritis

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.

Thrombus Permeability in Admission Computed Tomographic Imaging Indicates Stroke Pathogenesis Based on Thrombus Histology.

Background and Purpose- Intracranial thrombi can be characterized according to their permeability as measured by contrast agent penetration. Thrombus composition and its associated pathogenesis are important factors affecting treatment and secondary prevention. We aimed to explore the histopathologic factors explaining the heterogeneity of thrombus permeability measures and evaluated potential correlations with stroke pathogenesis. Methods- Thrombus densities were measured in thin-slice noncontrast computed tomography and automatically aligned computed tomographic angiography images of 133 patients with large-vessel occlusions of the middle cerebral artery. Change in thrombus attenuation (Δt) and corrected void fraction (ε; attenuation increase corrected for contralateral artery densities) were calculated. First, these thrombus perviousness measures were correlated with histological thrombus components (especially fractions of fibrin-platelet accumulation and red blood cells) and stroke pathogenesis (n=32). For validation, an association between perviousness and pathogenesis was assessed in a second, independent cohort (n=101). Results- Thrombus perviousness estimates were correlated with both fibrin/platelets fractions (Δt: r=0.43, P=0.016/ε: r=0.45, P=0.01) and inversely with red blood cells counts (Δt: r=-0.46, P=0.01/ε: r=-0.49, P=0.006). In the first cohort, Δt was substantially higher in samples from patients with cardioembolic stroke pathogenesis as compared with noncardioembolic-derived thrombi ( P=0.026). In the validation cohort, thrombus perviousness measures differed significantly between cardioembolic (Δt: median [ interquartile range]=12.53 [8.70-17.90]; ε: median [interquartile range]=0.054 [0.036-0.082]) and noncardioembolic thrombi (Δt: median [interquartile range]=3.2 [2.17-6.44], P<0.001; ε: median [interquartile range]=0.020 [0.011-0.027], P<0.001) and were associated with pathogenesis (Δt: β=0.45, P=0.016/ε: β=83.6, P=0.013) in a binary logistic regression model. Conclusions- Permeable thrombi showed a strong correlation with lower fractions of red blood cells counts and more fibrin/platelets conglomerations, concurrent with an association with cardioembolic origin. This novel information about thrombus perviousness may be valuable as a new and simple to acquire imaging marker for identifying stroke pathogenesis using early and readily available imaging.

Advanced age results in a diminished endothelial glycocalyx.

Age-related microvascular dysfunction is well characterized in rodents and humans, but little is known about the properties of the microvascular endothelial glycocalyx in advanced age. We examined the glycocalyx in microvessels of young and old male C57BL6 mice (young: 6.1 ± 0.1 mo vs. old: 24.6 ± 0.2 mo) using intravital microscopy and transmission electron microscopy and in human participants (young: 29 ± 1 yr vs. old: 60 ± 2 yr) using intravital microscopy. Glycocalyx thickness in mesenteric and skeletal muscle microvessels was 51-54% lower in old compared with young mice. We also observed 33% lower glycocalyx thickness in the sublingual microcirculation of humans in advanced age. The perfused boundary region, a marker of glycocalyx barrier function, was also obtained using an automated capture and analysis system. In advanced age, we observed a 10-22% greater perfused boundary region in mice and humans, indicating a more penetrable glycocalyx. Finally, using this automated analysis system, we examined perfused microvascular density and red blood cell (RBC) fraction. Perfused microvascular density is a marker of microvascular function that reflects the length of perfused microvessel segments in a given area; RBC fraction represents the heterogeneity in RBC presence between microvessel segments. Compared with young, the perfused microvascular density was 16-21% lower and RBC fraction was 5-14% lower in older mice and in older humans. These data provide novel evidence that, across mammalian species, a diminished glycocalyx is present in advanced age and is accompanied by markers of impaired microvascular perfusion. Age-related glycocalyx deterioration may be an important contributor to microvascular dysfunction in older adults and subsequent pathophysiology. NEW & NOTEWORTHY Advanced age is characterized by microvascular dysfunction that contributes to age-related cardiovascular diseases, but little is known about endothelial glycocalyx properties in advanced age. This study reveals, for the first time, lower glycocalyx thickness and barrier function that is accompanied by impaired microvascular perfusion in both mice and humans in advanced age.

Fractionation of Human Red Blood Cells Based on Intrinsic Magnetization

Fractionation of Human Red Blood Cells Based on Intrinsic Magnetization

Analysis of Hematocrit Variation Induced by Malaria-infected Red Blood Cells

The rheological properties of red blood cells (RBCs) in microvessels have a significant impact on the flow and transport of oxygen and nutrients in the blood. This paper used numerical methods to simulate the rheological property of the malaria-infected cells in microvessel, and compare the simulation results with other people’s findings. The results illustrate that in the 10-μm microvessel with low hematocrit (Hct) condition (18% and 27%), the Plasmodium falciparum-infected red blood cells (Pf-IRBCs) and healthy red blood cells (HRBCs) first form a “train”. In addition, the velocity of the Pf-IRBCs in the microvascular flow is slower than HRBCs, resulting in the increase of the surrounding erythrocyte hematocrit to about 40% or more. But this increase of volume fraction of RBCs is just temporary. It is observed that a similar phenomenon happens at the downstream region with 45%-Hct in the same microvessel. In the 20-μm microvessel with high-Hct condition (45%), the Pf-IRBCs slow down the velocity of the surrounding HRBCs and then locally elevate the volume fraction and result in the accumulating of the RBCs at the center of the vessels. Moreover, the Pf-IRBCs have a tendency to migrate to the edge of the RBC aggregates, which is called margination of red blood cells. This study is of some significance to the development of biomechanical experiments and devices.

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.