Ursula Windberger

Nature equipped red blood cells (RBCs) with diverse mechanical properties, which makes it possible to examine blood with different RBC properties (size, shape, aggregability, deformability). We investigated whether the shelf life of cow blood (stiff RBCs, low aggregability) is longer compared with pig blood (deformability/aggregability comparable to human) due to a delay in RBC clustering and decomposition. Blood was drawn from conscious pigs and cows in their familiar environment to reduce stress and stored 30 days at +7 °C. RBCs remained intact in cow samples whereas pig samples became hemolytic after day 20. White blood cells and platelets decreased with similar percentages in both species. Hematocrit (HCT) decreased due to RBC shrinking in bovine samples and due to RBC decay in porcine samples. Blood viscosity increased in both species although HCT decreased. In porcine samples, shear thinning decreased progressively, indicating a gradual loss of sample cohesion with storage. Yield stress and storage modulus decreased with hemolysis. In HCT-native cow samples, shear thinning, yield stress, and storage modulus showed high intraindividual variability, but the mean values did not change over the time course. In HCT-adjusted (38%) cow samples, solidification occurred after day 7, followed by a reduction in cohesion and shear thinning until the end of storage.

Nature provides red blood cell types (RBCs) with diverse mechanical properties, making possible to examine native blood in the presence of different RBC properties (size, shape, aggregability, deformability) without chemically changing the cell membranes, and without modifying the surrounding plasma. We investigated whether the shelf life of cow blood (stiff RBCs, low aggregability) is longer compared to pig blood (deformability/aggregability comparable to human) during cold storage due to a delay in RBC clustering and decomposition. Blood was drawn from conscious pigs and cows (CPDA-1) in their familiar environment to reduce stress, and stored 30 days at +7°C. RBCs remained intact in cow samples whereas pig samples became hemolytic after day 20. White blood cells and platelets decreased with similar percentage in both species. Hematocrit (HCT) decreased due to RBC shrinking in bovine, and due to RBC decay in porcine samples. Blood viscosity increased in both species although HCT decreased. In porcine samples shear-thinning decreased progressively, indicating gradual loss of sample cohesion with storage time. Also, yield stress and storage modulus decreased with hemolysis. In cow samples shear-thinning, yield stress, and storage modulus showed high intraindividual variability, but the mean values did not change over the time course.

BACKGROUND: Yielding and shear elasticity of blood are merely discussed within the context of hematocrit and erythrocyte aggregation. However, plasma might play a substantial role due its own viscoelasticity. OBJECTIVE: If only erythrocyte aggregation and hematocrit would determine yielding, blood of different species with comparable values would present comparable yield stresses. METHODS: rheometry (SAOS: amplitude and frequency sweep tests; flow curves) of hematocrit-matched samples at 37°C. Brillouin Light Scattering Spectroscopy at 38°C. RESULTS: Yield stress for pig: 20mPa, rat: 18mPa, and human blood: 9mPa. Cow and sheep blood were not in quasi-stationary state supporting the role of erythrocyte aggregation for the development of elasticity and yielding. However, pig and human erythrocytes feature similar aggregability, but yield stress of porcine blood was double. Murine and ruminant erythrocytes both rarely aggregate, but their blood behavior was fundamentally different. Pig plasma was shear-thinning and murine plasma was platelet-enriched, supporting the role of plasma for triggering collective effects and gel-like properties. CONCLUSIONS: Blood behavior near zero shear flow is not based solely on erythrocyte aggregation and hematocrit, but includes the hydrodynamic interaction with plasma. The shear stress required to break down elasticity is not the critical shear stress for dispersing erythrocyte aggregates, but the shear stress required to fracture the entire assembly of blood cells within their intimate embedding.

The cover image is based on the Research Article A Bayesian approach to blood rheological uncertainties in aortic hemodynamics by Sascha Ranftl et al., https://doi.org/10.1002/cnm.3576. Image credit: Sascha Ranftl, Julia Maderbacher, Thomas Stephan Müller, Wolfgang von der Linden, Ursula Windberger, and Günter Brenn. image

Results of an experimental study of blood properties are reported. To approach the boundary conditions of blood biophysics in a real flow, the viscoelastic measurements are carried out on surfaces coated with a 10-nm thick fibrous layer composed of extracellular matrix protein. For native whole blood, a plateau of the shear elastic modulus as a function of frequency is observed. The immediate consequence of the shear elasticity is that it is necessary to exceed the stress threshold so that blood can flow. This elastic threshold depends on the boundary conditions.

Given the use of modified blood products (e.g. leucocyte depleted erythrocyte concentrates in SAG-mannitol, dehydrated blood powder, defibrinated blood), drawing samples from conscious animals while minimizing stress should be considered a superior option in forensics. Nevertheless, the blood portion must be well described since individual differences in quality can occur, and storage will influence blood components qualitatively and quantitatively. Cow has been discussed as a suitable source of blood supply, but current data lack hematological and full rheological perspectives. Applying our own protocol for blood withdrawal in pigs (A. Sparer et al., Forensic Science International 2020;311:110268), this project includes the respective parameters in combination with passive drip pattern experiments during refrigerated storage in multiple study arms. Cow blood displayed a constant increase in viscosity (at high shear rate: 1000 s-1), reflecting the expected reduction in red blood cell (RBC) flexibility. RBCs shrank but remained intact with very few irregular shapes, therefore there was no evidence of hemolysis. Influence of storage on stain size in passive drip pattern experiments with different substrates was minimal. However in cows, it is not hemolysis but an early change in suspension properties that indicates storage lesion. Viscosity (at low shear rate: 1 s-1) of some blood samples increased three-fold (peaking at day 14), transitioning sharply to near-Newtonian (almost shear-independent) behavior thereafter. The higher this increase in viscosity, the greater the increase in the number of satellite spatter on glass. In order to ensure high quality simulations in the future, comprehensive rheological analyses to detect gradual changes in blood pseudoplasticity should be implemented in BPA.

Rats impress by their high platelet count resulting in hypercoagulability, which protects the animals from severe bleeding. However, platelets also import numerous stiff junction points into the fibrous system of a clot, also enhancing the pre-stress of the fibrin fibers, which lowers their deformability. Clot deformation is clinically important since large strains are present in the arterial tree (caused by the propagation of pressure and pulse waves), and a clot is considered “safe” when it can deform over a long range of strain amplitudes. We tested clot formation and the behavior of fully formed blood clots of laboratory rats at large sinusoidal shear stress amplitudes by rheometry and compared outcomes to human reference data. We found that fiber density (by scanning electron microscopy) and clot stiffness (by rheometry) was pronounced compared to humans and differed with sexual dimorphism and with rat strain. Using our large amplitude oscillation (LAOS) protocol, we detected that rat clots yielded with a frustrated attempt to stiffen instead of showing the macroscopic stiffening response that is typical for human clots. We attribute this behavior to the appearance of multiple microfractures until, finally, a few leading fibers uptake the load. Rat clots also failed to align fibers in shear direction to initiate affine deformation. The rat clot phenotype differs substantially from the human one, which must be considered in research and toxicological testing. If microfractures in the fiber meshwork are concentrated in vivo, parts of a clot may break off and be washed away. However, homogenously distributed microfractures may open pores and allow the penetration of plasminogen activators. What occurs in the rat vasculature depends on the on-site clot composition.

Blood plasma is not only a suspending medium for blood cells but exerts important further tasks in the vasculature. It couples RBC flow with its own flow, lubricates blood flow, and contributes to the endothelial and erythrocyte (RBC) surface layers. It was long believed exhibiting a pure Newtonian behaviour, but recent developments highlight an elastic nature that is reinforced at small scale. This surprising finding is attributed to careful dynamic mechanical tests optimizing the interfacial interactions between blood plasma and surfaces of the rheometry setup. In this frame, to approach the boundary conditions of blood biophysics in flow, we coated the surfaces with a hydrogel composed of extracellular matrix proteins to mimic a dissected aortic wall. We evidence for native whole blood a shear elastic plateau at about 30 mPa for frequencies within 0.3 and 0.6 rad s-1 and gap of 0.7 mm. Such behaviour indicates that the blood oscillates in the gap as a whole. We conclude that the slow flow of blood plasma observed close to the vessel wall can generate a stationary plasma layer that contributes to the functional width of surface layers in vessels. In endothelial cells, this layer uptakes the shear stress of flow first. The shear stress term calculated out of wall shear rate and blood viscosity becomes imprecise if it does not include this utmost important intermediate layer.