Pinar Ulker

Parameterization of red blood cell elongation index – shear stress curves obtained by ektacytometry

Abstract Measurement of red blood cell (RBC) deformability by ektacytometry yields a set of elongation indexes (EI) measured at various shear stresses (SS) presented as SS-EI curves, or tabulated data. These are useful for detailed analysis, but may not be appropriate when a simple comparison of a global parameter between groups is required. Based on the characteristic shape of SS-EI curves, two approaches have been proposed to calculate the maximal RBC elongation index (EImax) and the shear stress required for one-half of this maximal deformation (SS1/2): (i) linear Lineweaver-Burke (LB) model; (ii) Streekstra-Bronkhorst (SB) model. Both approaches have specific assumptions and thus may be subject to the measurement conditions. Using RBC treated with various concentrations of glutaraldehyde (GA) and data obtained by ektacytometry, the two approaches have been compared for nine different ranges of SS between 0.6–75 Pa. Our results indicate that: (i) the sensitivity of both models can be affected by the SS range and limits employed; (ii) over the entire range of SS-data, a non-linear curve fitting approach to the LB model gave more consistent results than a linear approach; (iii) the LB method is better for detecting SS1/2 differences between RBC treated with 0.001–0.005% glutaraldehyde (GA) and for a 40% mixture of rigid cells but is equally sensitive to SB for 10% rigid cells; and (iv) the LB and SB methods for EImax are equivalent for 0.001% and 0.003% GA and 40% rigid, with the SB better for 0.005% GA and the LB better for 10% rigid.

Mechanical stimulation of nitric oxide synthesizing mechanisms in erythrocytes

It has been previously demonstrated that red blood cells (RBC) possess functional nitric oxide (NO) synthesizing mechanisms. RBC are also equipped with variety of intracellular control mechanisms, and respond to mechanical forces and to various biological stimuli by increased release of ATP. Nitric oxide has also been demonstrated to be released from RBC under certain circumstances, and it has been hypothesized that NO synthase (NOS), which is located in both the RBC membrane and cytoplasm, might be activated by mechanical factors. The present study aimed at investigating NOS activation and NO export induced by mechanical stress applied to RBC in suspension. Heparinized venous blood samples were obtained from healthy, adult volunteers and their hematocrit adjusted to 0.4 l/l. The RBC suspensions were equilibrated at room temperature (22+/-2 degrees C) with either room air or made hypoxic (36 mmHg, approximately 70% saturation) using moisturized 100% nitrogen. The samples were then continuously pumped through a glass tube (diameter = 0.06 cm; length = 33 cm) for 30 min using a dual syringe pump to maintain a wall shear stress of 0.5-2 Pa with NO concentrations in the RBC suspensions measured electrochemically. NO concentration significantly increased under the influence of 2 Pa in hypoxic RBC suspensions: 105.0+/-14.2 nM to 127.1+/-12.0 nM as the peak value at 20 min of perfusion. No increase was observed at lower levels of shear stress. Plasma nitrite/nitrate concentrations were measured in samples obtained at five minute intervals. Application of fluid shear stress to hypoxic RBC suspensions resulted in a significant, time-dependent increase of plasma nitrite/nitrate levels, reaching to 14.7+/-1.5 microM from a control value of 11.2+/-1.3 microM. The presence of the non-specific NOS inhibitor L-NAME (10(-3) M) prevented this increment. Additionally, both eNOS and serine 1177 phosphorylated eNOS immuno-fluorescence staining in RBC cytoplasm were shown to increase in response to applied shear stress. Our results support the hypothesis that RBC NO synthase is activated and that export of NO from RBC is enhanced by mechanical stress.

Nitric oxide generation by endothelial cells exposed to shear stress in glass tubes perfused with red blood cell suspensions: role of aggregation

Endothelial function is modulated by wall shear stress acting on the vessel wall, which is determined by fluid velocity and the local viscosity near the vessel wall. Red blood cell (RBC) aggregation may affect the local viscosity by favoring axial migration. The aim of this study was to investigate the role of RBC aggregation, with or without altered plasma viscosity, in the mechanically induced nitric oxide (NO)-related mechanisms of endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured on the inner surface of cylindrical glass capillaries that were perfused with RBC suspensions having normal and increased aggregation at a nominal shear stress of 15 dyn/cm(2). RBC aggregation was enhanced by two different approaches: 1) poloxamer-coated RBC suspended in normal, autologous plasma, resulting in enhanced aggregation but unchanged plasma viscosity and 2) normal RBC suspended in autologous plasma containing 0.5% dextran (mol mass 500 kDa), with a similar level of RBC aggregation but higher plasma viscosity. Compared with normal cells in unmodified plasma, perfusion with suspensions of poloxamer-coated RBC in normal plasma resulted in decreased levels of NO metabolites and serine 1177 phosphorylation of endothelial nitric oxide synthase (eNOS). Perfusion with normal RBC in plasma containing dextran resulted in a NO level that remained elevated, whereas only a modest decrease of phosphorylated eNOS level was observed. The results of this study suggest that increases of RBC aggregation tendency affect endothelial cell functions by altering local blood composition, especially if the alterations of RBC aggregation are due to modified cellular properties and not to plasma composition changes.

Effects of storage duration and temperature of human blood on red cell deformability and aggregation

Blood samples used in hemorheological studies may be stored for a period of time, the effects of storage have yet to be fully explored. This study evaluated the effects of storage temperature (i.e., 4 degrees C or 25 degrees C) and duration on RBC deformability and aggregation for blood from healthy controls and from septic patients. Our results indicate that for normal blood, RBC deformability over 0.3-50 Pa is stable up to six hours regardless of storage temperature; at eight hours there were no significant differences in EI but SS1/2 calculated via a Lineweaver-Burk method indicated impaired deformability. Storage temperature affected the stable period for RBC aggregation: the safe time was shorter at 25 degrees C whereas at 4 degrees C aggregation was stable up to 12 hours. Interestingly, blood samples from septic patients were less affected by storage. Blood can thus be stored at 25 degrees C for up to six hours for deformability studies, but should be limited to four hours for RBC aggregation; storage at 4 degrees C may prolong the storage period up to 12 hours for aggregation but not deformability measurements. Therefore, the time period between sampling and measurement should be as short as possible and reported together with results.

Nitric oxide, erythrocytes and exercise

Nitric oxide (NO) is accepted to be an important factor affecting the degree of vascular tone in various portions of the circulation. Until recently, research in this area has focused on endothelial cells as a NO source, and there is general agreement that: 1) the level of wall shear stress is the primary determinant of endothelial nitric oxide synthase (eNOS) expression; 2) exercise training induces changes of endothelial cell NO synthesizing activity; 3) phosphorylation patterns of eNOS are altered following exercise episodes. However, there is now a growing body of evidence for the existence of similar nitric oxide synthesizing mechanisms in human red blood cells (RBC). Erythrocyte NOS activity has been demonstrated to be induced by applied shear stress and mechanical deformation of RBC, and there are closely linked increases of intracellular nitric oxide levels and of release of NO into the suspending phase. In brief, the RBC is an enzymatic source of NO that is dependent on flow dynamics and from which NO is released in very close proximity to vessel walls. Although reports regarding the influence of exercise on RBC nitric oxide synthesizing mechanisms are not yet concordant, it seems logical to suggest that this source of NO may play a role in the regulation of local blood flow dynamics during exercise.

Nitric oxide generated by red blood cells following exposure to shear stress dilates isolated small mesenteric arteries under hypoxic conditions

Red blood cells (RBC) possess a functional nitric oxide synthase (NOS) enzyme located in the cell membrane and cytoplasm. It has previously been observed that shear stress acting on RBC activates NOS and causes enhanced NO export. The aim of the present study was to investigate the physiological importance (e.g., in local blood flow regulation) of RBC-derived NO stimulated by application of shear stress. Blood samples and arterial vessel segments were obtained from Wistar rats; RBC suspensions were adjusted to a hematocrit of 0.1 l/l using Krebs solution. In order to apply shear stress to the RBC suspensions they were continuously flowed through a small-bore glass tube for 20 minutes at a wall shear stress of 2 Pa. The RBC suspensions were then perfused through endothelium denuded small mesenteric arteries having a diameter of ~300 μm under both high oxygen (PO2 ~130 mmHg) and hypoxic conditions. Perfusion of vessel segments with sheared RBC suspensions caused a significant dilation response under hypoxic conditions but not at high oxygen levels. Incubation of RBC suspensions with the non-specific NOS inhibitor L-NAME (10-3 M) prior to shear stress application abolished this dilation response. Our results indicate that NO released from RBC due to shear stress activation of NOS results in vasodilation of vessel segments under hypoxic conditions, and strongly suggest that NO originating from RBC may have a functional role in local blood flow regulation.

Nitric oxide generation in red blood cells induced by mechanical stress.

Previous reports have demonstrated that red blood cells (RBC) have an active nitric oxide (NO) synthesizing mechanism which has properties similar to endothelial nitric oxide synthase (eNOS). This red cell NOS activity contributes to the NO export from RBC. The present study explored the influence of shear stress applied to RBC on NO concentrations of cell suspensions. RBC were exposed to shear stress by filtration through 5 microm diameter pores under 10 cm H2O pressure, generating a wall shear stress of approximately 110Pa. NO concentration in the RBC suspensions were measured using electrochemical NO probes before and after filtration through the micropores. NO concentration was found to be significantly increased after a single passage of RBC suspensions through the micropores. The increment in NO concentration depended on the presence of calcium, being 21.8+/-4.4 nM with 1 mM calcium and 13.7+/-2.7 nM without added calcium. Including the calcium chelator EDTA completely abolished this increase. The increment of NO was also affected by the level of oxygenation, being more pronounced under hypoxic conditions. These results confirm that RBC NO generating mechanisms can be stimulated by exposing red cells to shear stress and that calcium plays a role in this stimulation.

Influence of tourniquet application on venous blood sampling for serum chemistry, hematological parameters, leukocyte activation and erythrocyte mechanical properties

Abstract Background: Venous blood sampling is usually performed using a tourniquet to help locate and define peripheral veins to achieve successful and safe venipuncture. Despite widespread usage of tourniquets for venipuncture by medical and laboratory staff, very few are aware of the effects of tourniquet application on laboratory parameters. In addition, definitive guidelines regarding when and how to use a tourniquet for blood sampling are lacking. The aim of the present study was to define the optimal sampling time after tourniquet removal to avoid adverse impact on laboratory analytes. Methods: Blood oxygen and carbon dioxide partial pressure, pH, oxyhemoglobin saturation (satO2), hematological parameters, serum electrolyte concentrations, erythrocyte, deformability and aggregation, leukocyte activation and nitrite/nitrate concentrations obtained 180 s after tourniquet release were compared with baseline values for 10 healthy subjects. Results: Blood gases, hematological parameters and serum electrolyte levels were not affected by the application and removal of a tourniquet. However, there were significant decreases in erythrocyte deformability at 90, 120, 180 s, and increases in erythrocyte aggregation at 5 and 30 s following removal of the tourniquet. A significant increase in granulocyte respiratory burst at 60 s was observed, confirming leukocyte activation due to application of the tourniquet. There were no significant alterations of blood nitrite/nitrate levels. Conclusions: Our blood sampling technique which mimicked the application and release of a tourniquet indicated unaltered values for routine blood gases, hematological testing and serum electrolyte levels. Conversely, hemorheological measurements can be affected. Therefore, it is strongly recommended that tourniquet application should be avoided during blood sampling or, if this is not possible, the procedure should be well standardized and details of the sampling method should be reported. Clin Chem Lab Med 2009;47:769–76.

Neither a Nitric Oxide Donor Nor Potassium Channel Blockage Inhibit RBC Mechanical Damage Induced by a Roller Pump

Red blood cells (RBC) are exposed to various levels of shear stresses when they are exposed to artificial flow environments, such as extracorporeal flow circuits and hemodialysis equipment. This mechanical trauma affects RBC and the resulting effect is determined by the magnitude of shear forces and exposure time. It has been previously demonstrated that nitric oxide (NO) donors and potassium channel blockers could prevent the sub-hemolytic damage to RBC, when they are exposed to 120 Pa shear stress in a Couette shearing system. This study aimed at testing the effectiveness of NO donor sodium nitroprussid (SNP, 10-4 M) and non-specific potassium channel blocker tetraethylammonium (TEA, 10-7 M) in preventing the mechanical damage to RBC in a simple flow system including a roller pump and a glass capillary of 0.12 cm diameter. RBC suspensions were pumped through the capillary by the roller pump at a flow rate that maintains 200 mmHg hydrostatic pressure at the entrance of the capillary. An aliquot of 10 ml of RBC suspension of 0.4 L/L hematocrit was re-circulated through the capillary for 30 minutes. Plasma hemoglobin concentrations were found to be significantly increased (~7 folds compared to control aliquot which was not pumped through the system) and neither SNP nor TEA prevented this hemolysis. Alternatively, RBC deformability assessed by laser diffraction ektacytometry was not altered after 30 min of pumping and both SNP and TEA had no effect on this parameter. The results of this study indicated that, in contrast with the findings in RBC exposed to a well-defined magnitude of shear stress in a Couette shearing system, the mechanical damage induced by a roller pump could not be prevented by NO donor or potassium channel blocker.

Effect of magnesium supplementation on blood rheology in NOS inhibition-induced hypertension model

This study investigated the effects of magnesium on blood rheological properties and blood pressure in nitric oxide synthase (NOS) inhibition-induced hypertension model. Hypertension was induced by oral administration of the nonselective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg/day) for 6 weeks and systolic blood pressure was measured by the tail-cuff method. The groups receiving magnesium supplementation were fed with rat chow containing 0.8% magnesium oxide during the experiment. At the end of experiment, blood samples were obtained from abdominal aorta, using ether anesthesia. Plasma and erythrocyte magnesium levels were determined by the atomic absorption spectrometer. RBC deformability and aggregation were determined by rotational ektacytometry. Plasma fibrinogen concentration was evaluated by ELISA. Whole blood and plasma viscosities were determined by viscometer and intracellular free Ca++ level was measured by using spectroflurometric method. Blood pressure was elevated in hypertensive groups and suppressed by magnesium therapy. Plasma viscosity and RBC aggregation were found to be higher in hypertensive rats than control animals and these parameters significantly decreased in magnesium supplemented hypertensive animals. Other measurements were not different between experimental groups. These results confirm that blood pressure, plasma viscosity and RBC aggregation increased in NOS inhibition-induced hypertension model and oral magnesium supplementation improved these parameters.