Our purpose was to identify the kinetics of erythrocyte (RBC) reversible aggregation (Agg) during stop flow (SF) experiments as a function of temperature and O2 for RBC from healthy subjects self‐identified as Black (B, n=8), Hispanic (H, 4) or Caucasian (C, 4) (48+/‐ 9 y). Whole blood (EDTA, BioIVT, Westbury, NY), vendor verified to be viral free, and used within 1 week of donation, was centrifuged and the platelet poor plasma (PPP) was recovered. All RBCs were resuspended in autologous PPP, and incubated at specified temperatures (37, 41, 45, 49 C) and O2 (0, 5, 10%, saturating). Flow was initiated in a microchannel system at 0.1‐0.2 Re, then stopped while recording the entire microchannel width. The greyscale range of the images showed a significant Fahraeus effect with flow (cell free layer at wall). With SF, as Agg proceeded, the aggregates accumulated within the centerline of the microchannel, increasing the variability of the greyscale range from white at the microchannel wall to black in the centerline. The change in variability indicated kinetics of Agg that occurred over 3 min of recording. The primary data of greyscale vs time (custom software) was fit to a sigmoid (dose response) curve, where the fitted EC50 was the T1/2 for Agg and with the fitted slope (p) indicated the kinetics of Agg. At 37 C the T1/2 was 93 s in 0% O2, 75 s with venous (5%) or arterial (10%) and was not different by group. As temperature increased, the T1/2 became significantly faster in blood from B or H (to 65 s), but not C. Correlation between temperature and the fitted T1/2 were significant in B (slope, R2; ‐2.9, 0.92), H (‐2.7, 0.97) and C (‐1.1, 0.6), but the F‐test on the slope was significant for B (p=0.02) and H (0.04) not C (0.6). This coincided with a significantly steeper p for B and H. At venous, arterial or saturating O2, the effect of increasing temperature to decrease the T1/2 was much less pronounced, and not different between groups. This data shows that in only hypoxic conditions, with temperatures of 41 C (105.8 F) or higher, RBC from self‐identified B or H subjects aggregates more readily than C. These results pertain to our focus of microvascular compromise in thermal burn injury progression, but also clinical conditions of localized cautery to control bleeding, high fevers or malignant hyperthermia.
Background Erythrocyte aggregation is a phenomenon that is commonly found in several pathological disease states: stroke, myocardial infarction, thermal burn injury, and COVID‐19. Erythrocyte aggregation is characterized by rouleaux, closely packed stacks of cells, forming three‐dimensional structures. Healthy blood flow monodisperses the red blood cells (RBCs) throughout the vasculature; however, in select pathological conditions, involving hyperthermia and hypoxemia, rouleaux formation remains and results in occlusion of microvessels with decreased perfusion. Objectives Our objective is to address the kinetics of rouleaux formation with sudden cessation of flow in variable temperature and oxygen conditions. Methods RBCs used in this in vitro system were obtained from healthy human donors. Using a vertical stop‐flow system aligned with a microscope, images were acquired and analyzed for increased variation in grayscale to indicate increased aggregation. The onset of aggregation after sudden cessation of flow was determined at proscribed temperatures (37–49°C) and oxygen (0%, 10%), and in the presence and absence of 4, 4′‐Diisothiocyano‐2,2′‐stilbenedisulfonic acid (DIDS). Both autologous and homologous plasma were tested. Results RBCs in autologous plasma aggregate faster and with a higher magnitude with both hyperthermia and hypoxemia. Preventing deoxyhemoglobin from binding to band 3 with DIDS (dissociates the cytoskeleton from the membrane) fully blocks aggregation. Further, RBC aggregation magnitude is greater in autologous plasma. Conclusions We show that the C‐terminal domain of band 3 plays a pivotal role in RBC aggregation. Further, aggregation is enhanced by hyperthermia and hypoxemia.
Despite their wide clinical usage, stent functionality may be compromised by complications at the site of implantation, including early/late stent thrombosis and occlusion. Although several studies have described the effect of fluid-structure interaction on local haemodynamics, there is yet limited information on the effect of the stent presence on specific hemorheological parameters. The current work investigates the red blood cell (RBC) mechanical behavior and physiological changes as a result of flow through stented vessels. Blood samples from healthy volunteers were prepared as RBC suspensions in plasma and in phosphate buffer saline at 45% haematocrit. Self-expanding nitinol stents were inserted in clear perfluoroalkoxy alkane tubing which was connected to a syringe, and integrated in a syringe pump. The samples were tested at flow rates of 17.5, 35 and 70 ml/min, and control tests were performed in non-stented vessels. For each flow rate, the sample viscosity, RBC aggregation and deformability, and RBC lysis were estimated. The results indicate that the presence of a stent in a vessel has an influence on the hemorheological characteristics of blood. The viscosity of all samples increases slightly with the increase of the flow rate and exposure. RBC aggregation and elongation index (EI) decrease as the flow rate and exposure increases. RBC lysis for the extreme cases is evident. The results indicate that the stresses developed in the stent area for the extreme conditions could be sufficiently high to influence the integrity of the RBC membrane.
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