Sublethal Supraphysiological Shear Stress Alters Erythrocyte Dynamics in Subsequent Low-Shear Flows

Abstract

Blood is a non-Newtonian, shear-thinning fluid owing to the physical properties and behaviors of red blood cells (RBCs). Under increased shear flow, pre-existing clusters of cells disaggregate, orientate with flow, and deform. These essential processes enhance fluidity of blood, although accumulating evidence suggests that sublethal blood trauma-induced by supraphysiological shear exposure-paradoxically increases the deformability of RBCs when examined under low-shear conditions, despite obvious decrement of cellular deformation at moderate-to-higher shear stresses. Some propose that rather than actual enhancement of cell mechanics, these observations are "pseudoimprovements" and possibly reflect altered flow and/or cell orientation, leading to methodological artifacts, although direct evidence is lacking. This study thus sought to explore RBC mechanical responses in shear flow using purpose-built laser diffractometry in tandem with direct optical visualization to address this problem. Freshly collected RBCs were exposed to a mechanical stimulus known to drastically alter cell deformability (i.e., prior shear exposure (PSE) to 100 Pa × 300 s). Samples were subsequently transferred to a custom-built slit-flow chamber that combined laser diffractometry with direct cell visualization. Cell suspensions were sheared in a stepwise manner (between 0.3 and 5.0 Pa), with each step being maintained for 15 s. Deformability and cell orientation indices were recorded for small-scatter Fraunhofer diffraction patterns and also visualized RBCs. PSE RBCs had significantly decreased visualized and laser-derived deformability at any given shear stress ≥1 Pa. Novel, to our knowledge, observations demonstrated that PSE RBCs had increased heterogeneity of direct visualized orientation with flow vector at any shear, which may be due to greater vorticity and thus instability in 5-Pa flow compared with unsheared control. These findings indicate that shear exposure and stress-strain history can alter subsequent RBC behavior in physiologically relevant low-shear flows. These findings may yield insight into microvascular disorders in recipients of mechanical circulatory support and individuals with hematological diseases that alter physical properties of blood.

Figure 1

Parametrization of red blood cell (RBC) deformability elongation index (EI)-shear curves from laser diffractometry or ektacytometry.

Figure 2

Design schematic for the custom-built, combined slit-flow ektacytometer-rheometer (i.e., the “ektacytoscope”). The designed system facilitated a coaxial assessment of blood cells in shear flow with high-speed visualization and small-scatter laser diffractometry. To see this figure in color, go online.

Figure 3

ImageJ (National Institutes of Health) image analysis process for the analysis of raw laser-diffractometry data and directly recorded visualized blood samples.

Figure 4

Typical images obtained from the ektacytoscope displaying captured laser-diffraction patterns and simultaneously recorded visualized RBCs at four increasing shear magnitudes. To see this figure in color, go online.

Figure 5

Deformability index of fresh RBC suspensions exposed to varied magnitude and duration of nonlethal shear stress. Increases in SS_1/2/EI_max reflect decreases in deformability. ^∗p < 0.05, significantly different to unsheared “0” sample.

Figure 6

(A) Ektacytometry deformability curves for unsheared Con and RBC suspensions previously exposed to 100 Pa for various durations. (B) Shown is the relationship of the decreased RBC deformability index and the increased EI obtained from laser-diffraction patterns at 0.3 Pa; r = 0.97.

Figure 7

EIs derived from coaxial laser diffractometry (A) and concurrent direct visualization rheometry (B) for RBCs without (Con) or after (prior shear exposure (PSE)) exposure to 100 Pa. ^∗p < 0.05, significantly different to Con.

Figure 8

Frequency distribution histograms of the EI of visualized flowing RBCs at discrete magnitudes of instantaneous shear stresses for unsheared Con and for RBCs with PSE.

Figure 9

Frequency of RBCs at discrete orientations for RBCs without (Con) or after (PSE) exposure to 100 Pa in subsequent 0.3- (A and B) and 5-Pa (C and D) shear flows. ^∗p < 0.05, significantly different to Con.