Erythrocytes, also known as red blood cells (RBCs), are the most abundant type of cells in the human body. Their distribution in the microcirculation determines the oxygen delivery and solute transport to tissues. This process relies on the partitioning of RBCs at successive bifurcations throughout the microvascular network and it is known since the last century that RBCs partition disproportionately to the fractional blood flow rate, therefore leading to heterogeneity of the hematocrit (i.e. volume fraction of RBCs in blood) in micro-vessels. Usually, downstream of a microvascular bifurcation, the vessel branch with a higher fraction of blood flow receives an even higher fraction of RBC flux. However, both temporal and time-average deviations from this phase-separation law have been observed in recent works.

Amongst others, the specific shape and high flexibility of RBCs might cause them to linger, i.e. RBCs can temporarily reside near the bifurcation apex with diminished velocity. This is of critical importance for the cells which are travelling in the capillaries of our circulatory system, whose cross section is sometimes smaller than the erythrocytes diameter. In this presentation, we quantify how the microscopic behavior of RBC influences their partitioning, through combined in vivo experiments and in silico simulations. In vivo experiments are performed in dorsal skinfold chambers of Golden Syrian hamsters, while in silico simulations reproduced the in vivo geometries and are performed using an immersed-boundary-lattice-Boltzmann method.

We developed an approach to quantify the cell lingering at highly-confined capillary-level bifurcations and demonstrate that it correlates with deviations of the phase-separation process from established empirical predictions by Pries et al. More accurately, we demonstrate a linear correlation between the lingering amplitude and deviations from the classical model of erythrocytes partitioning from the literature, both in vivo and in silico. We also show that the lingering is more pronounced in bifurcations presenting a higher curvature at their stagnation point, i.e. the intersection of the bifurcation’s wall and the flow divider plane. This explains why some geometries present stronger lingering than others. Furthermore, we shed light on how the cell membrane rigidity can affect the lingering behavior of RBCs, e.g. rigid cells tend to linger less than softer ones. Taken together, RBC lingering is an important mechanism that should be considered when studying how abnormal RBC rigidity in diseases such as malaria and sickle-cell disease could hinder the microcirculatory blood flow or how the vascular networks are altered under pathological conditions (e.g. thrombosis, tumors, aneurysm).

Blood is a vital fluid that plays an important role in transporting necessary substances and metabolic products for the cells. It consists of about 55% plasma and 45% blood cells where red blood cell (RBC) is the most abundant. Aggregation of RBCs is reversible under normal conditions, but abnormal clusters of RBCs appear in some pathological situations, such as diabetes. The formation mechanism of these clusters is controversial nowadays, but it has been demonstrated that some high-molecular-weight molecules such as fibrinogen and dextran could promote this aggregation.

RBCs surface is covered by glycolipids and glycoproteins, which are collectively called glycocalyx. The alteration of glycocalyx can affect the function of RBCs and it has been reported that a correlation between the enhanced aggregation and the glycoprotein layer (glycocalyx) degradation of the RBCs membrane could be found in diabetes. Other research has also shown that the glycocalyx alteration due to enzyme activities enhanced RBCs aggregation under static conditions by using rat blood. Against this background, glycocalyx alternation is an important point to be studied in this research, especially how glycocalyx alteration can affect RBCs aggregation under flow.

Amylase is a digestive enzyme that plays a crucial role in breaking down polysaccharides. As RBCs are covered by glycocalyx, amylase definitely interacts with RBCs’ glycocalyx. We used pancreatic amylase at various concentrations (0 – 2000U/L) to cleave glycocalyx, mimicking the pathologies of RBCs. We first confirmed the evolution of glycocalyx brush density as a function of amylase concentration with the help of Alexa488-conjugated wheat germ agglutinin (a specific marker of glycocalyx) and confocal microscopy. The results in absence of flow showed that at high concentrations of amylase, the RBCs glycocalyx underwent degradation. Then the aggregation size and morphology were then studied with an inverted microscope equipped with a high-speed camera and a blue light source. These static situation results proved not only the number of RBCs per aggregate was affected by the degradation of glycocalyx but also their morphology was influenced at the concentration of 150kDa dextran in Phosphate Buffered Saline (PBS). The aggregation activities of RBCs under flow were later studied in an artificial polydimethylsiloxane (PDMS) microfluidic circuit with variable pressure difference. The results also showed that the amylase-treated RBCs had significantly more aggregated than non-treated RBCs in 150kDa dextran (15mg/mL in PBS). Cleavage of glycocalyx considerably affected RBCs clusters' size, morphologies, and stabilities under flow.

The mechanism by which amylase caused glycocalyx alteration induces RBCs aggregation is not yet fully understood, and further research is needed to elucidate the exact pathways involved. However, our findings provide valuable insights into the role of glycocalyx alteration in RBC aggregation in absence of flow or in presence of flow and also suggest that glycocalyx degradation by pancreatic amylase has a clear impact on aggregation.

Introduction

Red blood cells (RBCs) are the most abundant cell type in mammals. They are characterized by a high surface-to-volume ratio which gives them very high deformability. When immersed into the flow of blood plasma, this high deformability creates a fascinatingly rich and intricate dynamics due to the two-way coupling between cellular and fluid mechanics. Besides its physiological relevance, e.g. for oxygen delivery, observations of red blood cell shapes have recently been suggested as a tool for medical diagnosis [1, 2]. With some exceptions, typical experimental setups are however limited to 2D microscopic imaging which can represent a serious limitation given the complexity of 3D RBC shapes in blood flow. If properly validated, computational simulations can resolve this issue and provide a deeper understanding of RBC dynamics. Most simulation models which are currently in use, however, are based on experimental validations using static (e.g. optical tweezers) or steady-state properties. In this talk, I will present computational modeling of single RBCs in microchannels focussing specifically on dynamics and transient situations.

Methods

We use boundary-integral computer simulations for the flow computations in the Stokes regime. Membrane dynamics are computed using Skalak and Helfrich laws for shear/area elasticity and bending, respectively. Our models furthermore include the viscous dissipation inside the membrane.

Results

The two prototypical states of a red blood cell flowing in a rectangular microchannel are the croissant and the slipper state [3]. The former is a stationary shape flowing in the center of the channel. The latter is characterized by an off-center flow position and, most importantly, by a constant rotation of the membrane around the liquid interior.

In the first part of the talk, I will briefly introduce computer simulations together with experimental data from our collaboration partners demonstrating that the croissant/slipper dynamics is in fact bistable and that the final RBC shape depends on the initial position. The main focus will then be to develop computational simulations that can quantitatively reproduce the rotation frequency of the membrane in the slipper state. In the second part, I will focus on transient situations such as transitions from croissants to slippers [4] as well as RBCs passing through channels with varying geometries.

I will show that, in order to obtain a computational model which is consistent with this highly diverse range of experiments, it is essential to (i) include membrane viscosity and (ii) employ a higher-than-usual ratio of the RBC interior to that of the outer fluid. Our simulations thus allow a direct and quantitative estimation of RBC membrane viscosity.

[1] Kubánková, M. et al. Biophys J 120, 2838–2847 (2021)

[2] Recktenwald, S. M. et al. eLife 11, e81316 (2022)

[3] Guckenberger, A. et al. Soft Matter 14, 2032–2043 (2018)

[4] Recktenwald, S. M. et al. Biophys J 121, 23–36 (2021)

Sickle Cell Disease (SCD) is a highly prevalent and handicapping genetic disease characterized by hemolytic anemia and unpredictable vaso-occlusive crises (VOC). It is caused by sickle hemoglobin, which, upon deoxygenation and dehydration, polymerizes into fibers in the cytoplasm of red blood cells (RBCs), increasing their cytoplasmic viscosity and remodeling their cytoskeleton. These changes combine to decrease the RBC deformability, a factor involved in the pathophysiology of SCD, with a key role in clinical outcome and occurrence of VOC. Even with reoxygenation, sickle hemoglobin polymerization is only partially reversible. Therefore, the RBC deformability of a sickle patient is highly dispersed as circulating RBCs have undergone a variable number of desoxygenation /reoxygenation cycles. To date, the assessment of RBC deformability require sophisticated devices. A simple method, requiring only a few blood microliters, easy to implement, fast, sensitive to the different parameters governing RBC deformability, and valid for heterogeneous RBC populations is lacking.

We exploit the fact that the individual movement of an RBC under shear flow is an indicator of its deformability. By increasing the flow shear rate, the motion of an RBC suspended in a medium of 40 mPa.s viscosity gradually changes from a rigid-like flip-flopping to a wheel-like rolling motion before transiting, above a critical shear rate set by the cell deformability, to a stationary droplet-like tank-treading motion. The more deformable the cell, the lower the critical shear rate. A given shear rate defines an RBC deformability threshold above which the cell is deformable enough to tanktread. We measured the fraction, f_TT, of tanktreading RBCs in a blood sample, which is the fraction of cells having a deformability higher than this threshold. The lower the applied shear rate, the more severe the deformability test and the fewer RBCs in tanktreading motion is observed in a sample; f_TT is a marker of deformability of the whole RBC sample, which can be tuned by the applied shear rate. We show that this marker is lower in SCD than in control samples. Furthermore, we show that in vitro, this parameter is sensitive to RBC density and hydration, two common features of SCD. We performed a clinical trial on 21 SCD patients by performing weekly repeated f_TT measures over a period of 7 to 23 weeks. We show that f_TT correlates with quantitative biological markers such as level of fetal hemoglobin, reticulocytes count, and plasma LDH level in SCD patients, thus suggesting that a higher hemolysis rate is associated with a lower RBC deformability; f_TT significantly decreases when the patient undergoes a high level of pain, and, surprisingly is increased when patients have an antihypertensor treatment. Moreover, f_TT significantly decreases before VOC onset and arrival at the hospital, then gradually increases during resolution of the VOC. Overall, f_TT could be useful for clinical monitoring of SCD patient to assess treatment efficacy or predict VOC. f_TT is also a good candidate to be an effective marker for diseases associated with RBC stiffening such as malaria.