Physical mechanisms of red blood cell splenic filtration.

The splenic interendothelial slits fulfill the essential function of continuously filtering red blood cells (RBCs) from the bloodstream to eliminate abnormal and aged cells. To date, the process by which 8 [Formula: see text]m RBCs pass through 0.3 [Formula: see text]m-wide slits remains enigmatic. Does the slit caliber increase during RBC passage as sometimes suggested? Here, we elucidated the mechanisms that govern the RBC retention or passage dynamics in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with submicron-wide physiologically calibrated slits. We observed that healthy RBCs pass through 0.28 [Formula: see text]m-wide rigid slits at 37 °C. To achieve this feat, 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, the cells with a low surface area-to-volume ratio (28% of RBCs in a 0.4 [Formula: see text]m-wide slit) must locally unfold their spectrin cytoskeleton inside the slit. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through the slits follows a [Formula: see text]1 and [Formula: see text]3 power law with in-slit pressure drop and slip width, respectively. This law is similar to that of a Newtonian fluid in a two-dimensional Poiseuille flow, showing that the dynamics of RBCs is controlled by their cytoplasmic viscosity. Altogether, our results 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.

Influence of storage and buffer composition on the mechanical behavior of flowing red blood cells.

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, which directly influence pressure regulation, oxygen perfusion, or platelet and white cell segregation toward 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 7 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 3 days after sample collection.

Acanthocyte Sedimentation Rate as a Diagnostic Biomarker for Neuroacanthocytosis Syndromes: Experimental Evidence and Physical Justification.

(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.

Vortical flow structures induced by red blood cells in capillaries.

OBJECTIVE: 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 conducted an experimental study on the flow field around red blood cells in capillary flow that is complemented by simulations of vortical flow between red blood cells. METHODS: Red blood cells were injected in a 10 × 12 µm rectangular microchannel at a low hematocrit, and the flow field around one or two cells was captured by a high-speed camera that tracked 250 nm nanoparticles in the flow field, acting as tracers. RESULTS: While the flow field around a steady "croissant" shape is found to be 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 complemented by numerical simulations.

Dextran adsorption onto red blood cells revisited: single cell quantification by laser tweezers combined with microfluidics.

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.

Effect of spectrin network elasticity on the shapes of erythrocyte doublets.

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.