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International audience; On-chip study of blood flow has emerged as a powerful tool to assess the contribution of each component of blood to its overall function. Blood has indeed many functions, from gas and nutrient transport to immune response and thermal regulation. Red blood cells play a central role therein, in particular through their specific mechanical properties, that directly influence pressure regulation, oxygen perfusion, or platelet and white cells segregation towards endothelial walls. As the bloom of in-vitro studies has led to the apparition of various storage and sample preparation protocols, we address the question of the robustness of the results involving cell mechanical behavior against this diversity. The effects of three conservation media (EDTA, citrate and glucose-albumin-sodium-phosphate) and storage time on the red blood cell mechanical behavior are assessed under different flow conditions: cell deformability by ektacytometry, shape recovery of cells flowing out of a microfluidic constriction, and cell flipping dynamics under shear flow. The impact of buffer solutions (phosphate-buffered saline and density-matched suspension using iodixanol/Optiprep) are also studied by investigating individual cell flipping dynamics, relative viscosity of cell suspensions and cell structuration under Poiseuille flow. Our results reveal that storing blood samples up to seven days after withdrawal and suspending them in adequate density-matched buffer solutions has in most experiments a moderate effect on the overall mechanical response, with a possible rapid evolution in the first three days after sample collection. SIGNIFICANCE Blood is in intimate contact with all organs in the body, supplying oxygen, nutrients and drugs while removing waste. It carries cells involved in immune response, wound repair and tumor dissemination. Blood is easily collected, revealing the presence of disease through biomarker analysis. It is storable and transfusable. Thus, blood is the subject of many in-vitro studies for research and medical purposes. Guidelines associated to sample preparation or storage conditions have been established, but these may affect its mechanical behavior. In this collaborative study, we provide new guidelines to minimize the impact of specific experimental requirements (e.g. density matching, blood freshness) by focusing on the single or collective motion of red blood cells in a large range of flow conditions.

Interendothelial slits in the spleen fulfill the major physiological function of continuously filtering red blood cells (RBCs) from the bloodstream to remove abnormal and aged cells. To date, the process of passage of 8 μm RBCs through 0.3-μm wide slits remains enigmatic. Should the slits increase their caliber during RBC passage as sometimes proposed in the literature? Here, we elucidated the mechanisms that govern the passage dynamics or retention of RBCs in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with slits of defined physiological dimensions, including submicron width. We observed that healthy RBCs pass through 0.28-μm wide rigid slits at body temperature. To achieve this tour de force, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, they must be able to locally unfold their spectrin cytoskeleton inside the slits. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through slits scales with in-slit pressure drop and slit width to the −1 and −3 power, respectively. This transit dynamics is similar to that of a Newtonian fluid in a 2D Poiseuille flow, thus showing that it is controlled by the RBC cytoplasmic viscosity. Altogether, our results clearly show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in-vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery.Significance StatementSplenic filtration of red blood cells through narrow interendothelial slits remains poorly understood despite its physiological significance as experiments and imaging of red cells passing through the slits are lacking. Here, we coupled live imaging, biomimetic submicron-fluidics, and multiscale modeling to quantify passage conditions. Remarkably, healthy 8-μm cells can pass through 0.28-μm slits at body temperature. This event is conditioned to cells being able to deform into two tether-connected equal spheres and to unfold their spectrin cytoskeleton. We showed that cells behave like a Newtonian fluid and that their dynamics is controlled by the inner fluid viscosity We thus propose an in-vitro and in-silico approach to quantify splenic clearance of diseased cells and cells engineered for transfusion and drug delivery

Knowledge about the flow field of the plasma around the red blood cells in capillary flow is important for a physical understanding of blood flow and the transport of micro- and nanoparticles and molecules in the flowing plasma. We conduct an experimental study on the flow field around red blood cells in capillary flow that are complemented by simulations of vortical flow between red blood cells. Red blood cells were injected in a 10x12 micrometer rectangular microchannel at a low hematocrit and the flow field around a single or two cells have been characterized thanks to a highspeed camera and by tracking 250 nm nanoparticles in flow behaving as tracers. While the flow field around a steady croissant shape is found to be relatively similar to that of a rigid sphere, the flow field around a slipper shape exhibits a small vortex at the rear of the red blood cell. Even more pronounced are vortex-like structures observed in the central region between two neighboring croissants. Conclusions: The rotation frequency of the vortices is to a good approximation, inversely proportional to the distance between the cells. Our experimental data are confirmed and complemented by numerical simulations., Preprint version of the manuscript that has been accepted for publication in Microcirculation

(1) Background: Chorea-acanthocytosis and McLeod syndrome are the core diseases among the group of rare neurodegenerative disorders called neuroacanthocytosis syndromes (NASs). NAS patients have a variable number of irregularly spiky erythrocytes, so-called acanthocytes. Their detection is a crucial but error-prone parameter in the diagnosis of NASs, often leading to misdiagnoses. (2) Methods: We measured the standard Westergren erythrocyte sedimentation rate (ESR) of various blood samples from NAS patients and healthy controls. Furthermore, we manipulated the ESR by swapping the erythrocytes and plasma of different individuals, as well as replacing plasma with dextran. These measurements were complemented by clinical laboratory data and single-cell adhesion force measurements. Additionally, we followed theoretical modeling approaches. (3) Results: We show that the acanthocyte sedimentation rate (ASR) with a two-hour read-out is significantly prolonged in chorea-acanthocytosis and McLeod syndrome without overlap compared to the ESR of the controls. Mechanistically, through modern colloidal physics, we show that acanthocyte aggregation and plasma fibrinogen levels slow down the sedimentation. Moreover, the inverse of ASR correlates with the number of acanthocytes (R2=0.61, p=0.004). (4) Conclusions: The ASR/ESR is a clear, robust and easily obtainable diagnostic marker. Independently of NASs, we also regard this study as a hallmark of the physical view of erythrocyte sedimentation by describing anticoagulated blood in stasis as a percolating gel, allowing the application of colloidal physics theory.

Chorea-acanthocytosis (ChAc) and McLeod syndrome (MLS) are the core diseases among the group of rare neurodegenerative disorders that comprise neuroacanthocytosis syndrome (NAS). Both ChAc and MLS patients present with an increased number of irregularly spiky erythrocytes, so-called acanthocytes. The detection of acanthocytes is often a crucial parameter in the diagnosis of NAS. However, this approach is error-prone and not very reliable, typically explaining the misdiagnosis of NAS patients. Based on the standard Westergren method, we show that compared with that in healthy controls, the erythrocyte sedimentation rate (ESR) with a two-hour read-out is significantly prolonged in ChAc and MLS with no overlap. Thus, the ESR is a clear, robust and easily obtained diagnostic marker. Mechanistically, by applying modern colloidal physics, we show that acanthocyte aggregation and plasma fibrinogen levels slow the sedimentation process. Apart from its diagnostic value, ESR may also be the first biomarker for monitoring treatments for NAS patients. Further studies are required to test whether the ESR may also detect other NASs. In addition to medical progress, this study is also a hallmark of the physical view of the erythrocyte sedimentation process by describing anticoagulated blood in stasis as a percolating gel, allowing the application of colloidal physics theory.

Red blood cell (RBC) aggregation highly influences hemorheology and blood microcirculation in the human body. The aggregation properties of RBCs can vary due to numerous factors, including RBC age. The aim of this work was to estimate in vitro the differences in the RBC aggregation properties of different RBC age populations in single-cell experiments using laser tweezers. RBCs from five healthy volunteers were separated into four subpopulations by Percoll density gradient centrifugation. Each subpopulation of the RBC was separately resuspended in autologous plasma or dextran 70 kDa (50 mg/mL). The aggregation force between the single cells was measured with holographic laser tweezers. The obtained data demonstrated an enhancement of RBC aggregation force in doublets with age: the older the cells, the higher the aggregation force. The obtained data revealed the differences between the aggregation and aggregability of RBC in dependence of the RBC in vivo age.

Red blood cell (RBC) aggregates play an important role in determining blood rheology. RBCs in plasma or polymer solution interact attractively to form various shapes of RBC doublets, where the attractive interactions can be varied by changing the solution conditions. A systematic numerical study on RBC doublet formation is performed, which takes into account the shear elasticity of the RBC membrane due to the spectrin cytoskeleton, in addition to the membrane bending rigidity. RBC membranes are modeled by two-dimensional triangular networks of linked vertices, which represent three-dimensional cell shapes. The phase space of RBC doublet shapes in a wide range of adhesion strengths, reduced volumes, and shear elasticities is obtained. The shear elasticity of the RBC membrane changes the doublet phases significantly. Experimental images of RBC doublets in different solutions show similar configurations. Furthermore, we show that rouleau formation is affected by the doublet structure.

The aggregation of red blood cells (RBC) is of importance for hemorheology, while its mechanism remains debatable. The key question is the role of the adsorption of macromolecules on RBC membranes, which may act as “bridges” between cells. It is especially important that dextran is considered to induce “bridge”-less aggregation due to the depletion forces. We revisit the dextran-RBC interaction on the single cell level using the laser tweezers combined with microfluidic technology and fluorescence microscopy. An immediate sorption of ~104 molecules of 70 kDa dextran per cell was observed. During the incubation of RBC with dextran, a gradual tenfold increase of adsorption was found, accompanied by a moderate change in the RBC deformability. The obtained data demonstrate that dextran sorption and incubation-induced changes of the membrane properties must be considered when studying RBC aggregation in vitro.