Erythrocyte morphological symmetry analysis to detect sublethal trauma in shear flow.

The viscoelastic properties of red blood cells (RBC) facilitate flexible shape change in response to extrinsic forces. Their viscoelasticity is intrinsically linked to physical properties of the cytosol, cytoskeleton, and membrane-all of which are highly sensitive to supraphysiological shear exposure. Given the need to minimise blood trauma within artificial organs, we observed RBC in supraphysiological shear through direct visualisation to gain understanding of processes leading to blood damage. Using a custom-built counter-rotating shear generator fit to a microscope, healthy red blood cells (RBC) were directly visualised during exposure to different levels of shear (10-60 Pa). To investigate RBC morphology in shear flow, we developed an image analysis method to quantify (a)symmetry of deforming ellipsoidal cells-following RBC identification and centroid detection, cell radius was determined for each angle around the circumference of the cell, and the resultant bimodal distribution (and thus RBC) was symmetrically compared. While traditional indices of RBC deformability (elongation index) remained unaltered in all shear conditions, following ~100 s of exposure to 60 Pa, the frequency of asymmetrical ellipses and RBC fragments/extracellular vesicles significantly increased. These findings indicate RBC structure is sensitive to shear history, where asymmetrical morphology may indicate sublethal blood damage in real-time shear flow.

Shear-stress mediated nitric oxide production within red blood cells: A dose-response.

BACKGROUND: Red blood cells (RBC) are exposed to varying shear stress while traversing the circulatory system; this shear initiates RBC-derived nitric oxide (NO) production. OBJECTIVE: The current study investigated the effect of varying shear stress dose on RBC-derived NO production. METHODS: Separated RBC were prepared with the molecular probe, diamino-fluoreoscein diacetate, for fluorometric detection of NO. Prepared RBC were exposed to discrete magnitudes of shear stress (1-100 Pa), and intracellular and extracellular fluorescence was quantified via fluorescence microscopy at baseline (0 min) and discrete time-points (1-30 min). RESULTS: Intracellular RBC-derived NO fluorescence was significantly increased (p < 0.05) following shear stress exposure when compared to baseline at: i) 1 min-100 Pa; ii) 5 min-1, 5 Pa; iii) 15 min-1, 5, 35 Pa; iv) 30 min-35 Pa. Extracellular RBC-derived NO fluorescence was significantly increased (p < 0.05) following shear stress exposure when compared to baseline at: i) 5 min - 100 Pa; ii) 15 min-100 Pa; iii) 30 min-40, 100 Pa. CONCLUSIONS: These data indicate that: i) a dose-response exists for the RBC-derived production of NO via shear stress; and ii) exposure to supra-physiological shear stress allows for the leakage of RBC intracellular contents (e.g., RBC-derived NO).

Visualization of erythrocyte deformation induced by supraphysiological shear stress.

To elucidate development of shear-induced damage in erythrocytes, it is necessary to visualize erythrocytes under high-shear flow. Therefore, we prototyped a special shear flow chamber with a counter-rotating mechanism consisting of a transparent acrylic cone and a glass plate. The flow chamber was mounted on an inverted microscope and illuminated by a 350-W metal halide lamp. This experimental system made it possible for a digital video camera to record through the microscopes' objective lens the rheological behavior in shear flow of erythrocytes diluted in highly viscous polyvinyl pyrrolidone. We successfully visualized the blood cells' ellipsoidal deformation response to an unphysiological, high shear stress of 288 Pa, their shift into abnormal rheological behavior, and final collapse. When abnormality first appeared, the membrane surface of some ellipsoidal erythrocytes started undulating and their shape became more asymmetric. Finally, the erythrocytes appeared to fragment, although the fragments continued tumbling together suggesting that they were all still connected. One such abnormal erythrocyte became segmented through collision with other cell. The undulation of the membrane surface when erythrocytes experienced trauma suggests possible detachment of the lipid bilayer from the membrane cytoskeleton. As the damage increased, the morphological abnormality of cells became greater with less tank-treading, and then, the erythrocytes started tumbling. This unstable behavior increases the volume of flow region occupied by the erythrocytes and increases the chance that neighboring cells will hit them and break them into segmented pieces. This study clearly showed that the beginning of erythrocytes' morphological abnormality was induced by shear stress.