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 and RBC-NOS Serine1177 Phosphorylation in Humans: A Dose Response.

Red blood cells (RBC) express a nitric oxide synthase isoform (RBC-NOS) that appears dependent on shear stress for Serine1177 phosphorylation. Whether this protein is equally activated by varied shears in the physiological range is less described. Here, we explored RBC-NOS Serine1177 phosphorylation in response to shear stress levels reflective of in vivo conditions. Whole blood samples were exposed to specific magnitudes of shear stress (0.5, 1.5, 4.5, 13.5 Pa) for discrete exposure times (1, 10, 30 min). Thereafter, RBC-NOS Serine1177 phosphorylation was measured utilising immunofluorescence labelling. Shear stress exposure at 0.5, 1.5, and 13.5 Pa significantly increased RBC-NOS Serine1177 phosphorylation following 1 min (p < 0.0001); exposure to 4.5 Pa had no effect after 1 min. RBC-NOS Serine1177 phosphorylation was significantly increased following 10 min at each magnitude of shear stress (0.5, 1.5, 13.5 Pa, p < 0.0001; 4.5 Pa, p = 0.0042). Shear stress exposure for 30 min significantly increased RBC-NOS Serine1177 phosphorylation at 0.5 Pa and 13.5 Pa (p < 0.0001). We found that RBC-NOS phosphorylation via shear stress is non-linear and differs for a given magnitude and duration of exposure. This study provides a new understanding of the discrete relation between RBC-NOS and shear stress.

Red blood cell tolerance to shear stress above and below the subhemolytic threshold.

Mechanical circulatory support device (MCS) design has improved over the years and yet blood damage (e.g., hemolysis) remains a problem. Accumulating evidence indicates a subhemolytic threshold for red blood cells (RBC)-a threshold at which RBC deformability is impaired prior to hemolysis. The current study aimed to assess the deformability of RBC exposed to supra-physiological shear stresses that are typical of MCS devices and assess whether a method used to estimate an individualized subhemolytic threshold, accurately demarcates whether future application of shear stress was damaging. Suspensions of RBC were "conditioned" with discrete magnitudes of shear stress (5-100 Pa) for specific durations (1-16 s). Cellular deformability was subsequently measured via ektacytometry and a mechanical sensitivity (MS) index was calculated to identify the subhemolytic threshold. Thereafter, fresh RBC suspensions were exposed to a magnitude of shear stress 10 Pa above, 10 Pa below, or matched to a donor's previously estimated subhemolytic threshold for a given duration (1, 4, 16 s) to ascertain the sensitivity of the subhemolytic threshold. The MS index of RBC was significantly impaired following exposure to 10 Pa above the subhemolytic threshold (p < 0.0001), and significantly enhanced following exposure to 10 Pa below the subhemolytic threshold (p < 0.01). For all shear conditions, there was no significant increase in free hemoglobin. Functional assessments of RBC may be useful when conducting biocompatibility testing of MCS devices, to detect trauma to blood prior to overt cell rupture being induced.

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).

Speed Modulation of the HeartWare HVAD to Assess In Vitro Hemocompatibility of Pulsatile and Continuous Flow Regimes in a Rotary Blood Pump.

Although rotary blood pumps (RBPs) sustain life, blood exposure to continuous supra-physiological shear stress induces adverse effects (e.g., thromboembolism); thus, pulsatile flow in RBPs represents a potential solution. The present study introduced pulsatile flow to the HeartWare HVAD using a custom-built controller and compared hemocompatibility biomarkers (i.e., platelet aggregation, concentrations for ADAMTS13, von Willebrand factor (vWf), and free-hemoglobin in plasma (pfHb), red blood cell (RBC) deformability, and RBC-nitric oxide synthase (NOS) activity) between continuous and pulsatile flow in a blood circulation loop over 5 h. The HeartWare HVAD was operated using a custom-built controller, at continuous speed (3282 rev/min) or in a pulsatile mode (mean speed = 3273 rev/min, amplitude = 430 rev/min, frequency = 1 Hz) to generate a blood flow rate of 5.0 L/min, HVAD differential pressure of 90 mm Hg for continuous flow and 92 mm Hg for pulsatile flow, and systolic and diastolic pressures of 121/80 mm Hg. For both flow regimes, the current study found; (i) ADP- and collagen-induced platelet aggregation, and ADAMTS13 concentration significantly decreased after 5 h (P < 0.01; P < 0.05), (ii) ristocetin-induced platelet aggregation significantly increased after 45 min (P < 0.05), (iii) vWf concentration did not significantly differ at any time point, (iv) pfHb significantly increased after 5 h (P < 0.01), (v) RBC deformability improved during the continuous flow regime (P < 0.05) but not during pulsatile flow, and (vi) RBC-NOS activity significantly increased during continuous flow (15 min), and pulsatile flow (5 h; P < 0.05). The current study demonstrated: (i) speed modulation does not improve hemocompatibility of the HeartWare HVAD based on no observable differences being detected for routine biomarkers, and (ii) the time-course for increased RBC-NOS activity observed during continuous flow may have improved RBC deformability.

Oxidative Stress Increases Erythrocyte Sensitivity to Shear-Mediated Damage.

Patients receiving mechanical circulatory support often present with heightened inflammation and free radical production associated with pre-existing conditions in addition to that which is due to blood interactions with nonbiological surfaces. The aim of this experimental laboratory study was to assess the deformability of red blood cells (RBC) previously exposed to oxygen free radicals and determine the susceptibility of these cells to mechanical forces. In the present study, RBC from 15 healthy donors were washed and incubated for 60 min at 37°C with 50 µM phenazine methosulfate (PMS; an agent that generates superoxide within RBC). Incubated RBC and negative controls were assessed for their deformability and susceptibility to mechanical damage (using ektacytometry) prior to the application of shear stress, and also following exposure to 25 different shear conditions of varied magnitudes (shear stress 1, 4, 16, 32, 64 Pa) and durations (1, 4, 16, 32, 64 s). The salient findings demonstrate that incubation with PMS impaired important indices of RBC deformability indicating altered cell mechanics by ∼19% in all conditions (pre- and postexposure to shear stress). The typical trends in shear-mediated changes in RBC susceptibility to mechanical damage, following conditioning shear stresses, were maintained for PMS incubated and control conditions. We demonstrated that free radicals hinder the ability of RBC to deform; however, RBC retained their typical mechanical response to shear stress, albeit at a decreased level compared with control following exposure to PMS. Our findings also indicate that low shear exposure may decrease cell sensitivity to mechanical damage upon subsequent shear stress exposures. As patients receiving mechanical circulatory support have elevated exposure to free radicals (which limits RBC deformability), concomitant exposure to high shear environments needs to be minimized.

Repetitive Supra-Physiological Shear Stress Impairs Red Blood Cell Deformability and Induces Hemolysis.

The supra-physiological shear stress that blood is exposed to while traversing mechanical circulatory assist devices affects the physical properties of red blood cells (RBCs), impairs RBC deformability, and may induce hemolysis. Previous studies exploring RBC damage following exposure to supra-physiological shear stress have employed durations exceeding clinical instrumentation, thus we explored changes in RBC deformability following exposure to shear stress below the reported "hemolytic threshold" using shear exposure durations per minute (i.e., duty-cycles) reflective of that employed by circulatory assist devices. Blood collected from 20 male donors, aged 18-38 years, was suspended in a viscous medium and exposed to an intermittent shear stress protocol of 1 s at 100 Pa, every 60 s for 60 duty-cycles. During the remaining 59 s/min, the cells were left at stasis until the subsequent duty-cycle commenced. At discrete time points (15/30/45/60 duty-cycles), an ektacytometer measured RBC deformability immediately after shear exposure at 100 Pa. Plasma-free hemoglobin, a measurement of hemolysis, was quantified via spectrophotometry. Supra-physiological shear stress impaired RBC properties, as indicated by: (1) decreased maximal elongation of RBCs at infinite shear stress following 15 duty-cycles (P <0.05); (2) increased real-time RBC deformability during application of the supra-physiological shear stress protocol (100 Pa) following exposure to 1 duty-cycle (F (1.891, 32.15) = 12.21, P = 0.0001); and (3) increased plasma-free hemoglobin following 60 duty-cycles (P < 0.01). The present study indicates that exposure of RBCs to short-term, repeated supra-physiological shear stress, impairs RBC deformability, with the extent of impairment exacerbated with each duty-cycle, and ultimately precipitates hemolysis.