Laser Ektacytometry Research Articles

Estimating of Fraction of Weakly Deformable Erythrocytes in a Blood Sample Based on the Method of Laser Ektacytometry and a Database of Simulated Diffraction Patterns

Estimating of Fraction of Weakly Deformable Erythrocytes in a Blood Sample Based on the Method of Laser Ektacytometry and a Database of Simulated Diffraction Patterns

Calibration Problem in Laser Ektacytometry of Erythrocytes

The problem of measuring the deformability of erythrocytes by laser diffractometry in shear flow (ektacytometry) is considered. The question of how the shape of the isointensity line in the diffraction pattern, which arises when a laser beam is scattered by an ensemble of erythrocytes, is related to the level of light intensity on this line, is analyzed. A simplified algorithm for measuring the parameters of the distribution of erythrocytes in deformability is proposed, which does not require photometry of the central part of the diffraction pattern.

Line curvature algorithm in laser ektacytometry of red blood cells

The iso-intensity line curvature algorithm in laser ektacytometry of red blood cells is investigated by numerical simulation. The algorithm is designed to measure the average deformability, as well as the width and asymmetry of the red blood cell deformability distribution in a blood sample under study. The accuracy and scope of the algorithm are determined. Using a bimodal ensemble as an example, the possibility of determining the fraction of weakly deformable red blood cells in a blood sample by laser ektacytometry is demonstrated.

Numerical reconstruction of two-dimensional particle size distributions from laser diffraction data

In this paper, we consider the two-dimensional Fredholm integral equation of the first kind. The kernel of this equation models the scattering of a laser beam by spheroids having the same fixed orientation. The unknown function under the integral describes the distribution of spheroids along two semi-axes. The input data are the diffraction pattern corresponding to the scattering of the laser beam by the particles. We show that the Tikhonov regularization method allows one to reconstruct two-dimensional distributions in the case when the diffraction pattern is modulated by white noise with relative amplitude up to 1%. In applications, this means novel possibility of obtaining two-dimensional particle size distributions, rather than one-dimensional ones, as in the classical version of the method. This significantly expands the capabilities of particle sizing via static laser diffraction technique. We show that the solution of the corresponding equation is unique in space and exists when the right-hand-side function is in a set dense in . We provide test experimental results in the framework of laser ektacytometry of red blood cells, which serves as the main application of the proposed approach presently.

The Effects of Different Signaling Pathways in Adenylyl Cyclase Stimulation on Red Blood Cells Deformability.

Signaling pathways of red blood cells’ (RBCs) micromechanics regulation, which are responsible for maintaining microcirculation, constitute an important property of RBC physiology. Selective control over these processes may serve as an indispensable tool for correction of hemorheological disorders, which accompany a number of systemic diseases (diabetes mellitus I&II, arterial hypertension, malaria, etc.). Activation of certain pathways involving adenylyl cyclase may provide fast adaptive regulation of RBC deformability (RBC-D). However the specific molecular conditions of intracellular signal transduction in mediating RBC microrheological properties at adenylyl cyclase stimulation remain unclear. In this paper, we present the results of the in vitro study of the effects of different signaling pathways in adenylyl cyclase stimulation on RBC-D. We studied (1) the direct stimulation of adenylyl cyclase with forskolin; (2) non-selective adrenoreceptor stimulation with epinephrine; (3) β2-adrenoreceptor agonist metaproterenol; (4) membrane-permeable analog of cAMP (dibutyryl-cAMP). Using laser ektacytometry, we observed a concentration-dependent increase in RBC-D for all studied effectors. The EC50 values for each substance were estimated to be in the range of 1–100 μM depending on the shear stress applied to the RBC suspension. The results can serve as an evidence of adenylyl cyclase signaling cascade involvement in the regulation of RBC micromechanical properties presenting a complex molecular pathway for fast increase of microcirculation efficiency in case of corresponding physiologic metabolic demands of the organism, e.g., during stress or physical activity. Further studies of this molecular system will reveal new knowledge which may improve the quality of medical treatment of hemorheological disorders.

Curvature of a line defined as a discrete set of points, and diffraction pattern processing for laser ektacytometry of red blood cells

The problem of measuring the erythrocyte deformability by laser diffractometry in a shear flow (ektacytometry) is considered. A procedure for processing diffraction patterns is proposed, which makes it possible to measure the curvature of the isointensity lines with high accuracy. The procedure involves finding the specific points of the isointensity line, which we called the band points. The coordinates of these points are determined by the isointensity line (band) width, depending on the resolution and dynamic range of the camera. Determining the coordinates of the band points allows us to calculate the curvature of the line by the formula of the second differential derivative. The accuracy of the procedure is justified with the help of analytical estimates of the second and fourth derivatives of the function describing the isointensity line. It is shown that to measure the curvature of the isointensity line with an error not exceeding one percent, it is necessary to photograph the neighborhoods of the points of interest to us with a resolution of about 10 megapixels or to take an image of the entire diffraction pattern with a resolution of 100 megapixels.

On the possibility of laser ektacytometry measurement of the kurtosis for erythrocyte deformability distribution

The problem of measuring red blood cell deformability by laser diffractometry in shear flow (ektacytometry) is considered. An algorithm has been developed for measuring the four central parameters of the erythrocyte deformability distribution, i.e. mean value, variance, asymmetry coefficient and kurtosis. The algorithm is designed to work with the peripheral part of the diffraction pattern and serves to analyse weakly inhomogeneous ensembles of red blood cells.

Characteristic point algorithm in laser ektacytometry of red blood cells

We consider the problem of measuring red blood cell deformability by laser diffractometry in shear flow (ektacytometry). A new equation is derived that relates the parameters of the diffraction pattern to the width of the erythrocyte deformability distribution. The numerical simulation method shows that this equation provides a higher accuracy of measurements in comparison with the analogous equation obtained by us earlier.

УЛУЧШЕННЫЙ АЛГОРИТМ ОБРАБОТКИ ДАННЫХ ДЛЯ ЛАЗЕРНОЙ ЭКТАЦИТОМЕТРИИ ЭРИТРОЦИТОВ

УЛУЧШЕННЫЙ АЛГОРИТМ ОБРАБОТКИ ДАННЫХ ДЛЯ ЛАЗЕРНОЙ ЭКТАЦИТОМЕТРИИ ЭРИТРОЦИТОВ

New diffractometric equations and data processing algorithm for laser ektacytometry of red blood cells

The problem of measuring the deformability of red blood cells in shear flow by laser diffractometry (ektacytometry) is considered. New diffractometric equations are obtained, which relate the parameters of the diffraction pattern to the characteristics of the erythrocyte ensemble, such as mean deformability as well as width and asymmetry of the erythrocytes distribution in deformability. A feature of these equations is that they include only geometric parameters of the diffraction pattern but do not contain its energy parameters. Basing on these equations, we propose a new data processing algorithm for the laser ektacytometry of red blood cells.

A simplified algorithm for measuring erythrocyte deformability dispersion by laser ektacytometry

The possibility of measuring the dispersion of red blood cell deformability by laser diffractometry in shear flow (ektacytometry) is analysed theoretically. A diffraction pattern parameter is found, which is sensitive to the dispersion of erythrocyte deformability and to a lesser extent – to such parameters as the level of the scattered light intensity, the shape of red blood cells, the concentration of red blood cells in the suspension, the geometric dimensions of the experimental setup, etc. A new algorithm is proposed for measuring erythrocyte deformability dispersion by using data of laser ektacytometry.

Study of laser beam scattering by inhomogeneous ensemble of red blood cells in a shear flow

Laser ektacytometry is a technique widely used for measuring the deformability of red blood cells (erythrocytes) in blood samples in vitro. In ektacytometer, a flow of highly diluted suspension of erythrocytes in variable shear stress conditions is illuminated with a laser beam to obtain a diffraction pattern. The diffraction pattern provides information about the shapes (shear-induced elongations) of the cells under investigation. This paper is dedicated to developing the technique of laser ektacytometry so that it would enable one to measure the distribution of the erythrocytes in deformability. We discuss the problem of calibration of laser ektacytometer and test a novel data processing algorithm allowing to determine the parameters of the distribution of erythrocytes deformability. Experimentally, we examined 12 specimens of blood of rats under the action of 4 shear stresses. Analysis of the data shows that in conditions of a limited range of digitizing the diffraction patterns, the measurement errors for the mean deformability, deformability scatter and the skewness of erythrocytes distribution in deformability by our method are respectively 15%, 20% and 20%.

Measuring skewness of red blood cell deformability distribution by laser ektacytometry

An algorithm is proposed for measuring the parameters of red blood cell deformability distribution based on laser diffractometry of red blood cells in shear flow (ektacytometry). The algorithm is tested on specially prepared samples of rat blood. In these experiments we succeeded in measuring the mean deformability, deformability variance and skewness of red blood cell deformability distribution with errors of 10%, 15% and 35%, respectively.

Laser ektacytometry and evaluation of statistical characteristics of inhomogeneous ensembles of red blood cells

The paper is devoted to development of the laser ektacytometry technique for evaluation of the statistical characteristics of inhomogeneous ensembles of red blood cells (RBCs). We have analyzed theoretically laser beam scattering by the inhomogeneous ensembles of elliptical discs, modeling red blood cells in the ektacytometer. The analysis shows that the laser ektacytometry technique allows for quantitative evaluation of such population characteristics of RBCs as the cells mean shape, the cells deformability variance and asymmetry of the cells distribution in the deformability. Moreover, we show that the deformability distribution itself can be retrieved by solving a specific Fredholm integral equation of the first kind. At this stage we do not take into account the scatter in the RBC sizes.

Effects of ethanol on red blood cell rheological behavior

Consumption of red wine is associated with a decreased risk of several cardiovascular diseases (e.g., coronary artery disease, stroke), but unfortunately literature reports regarding ethanol's effects on hemorheological parameters are not concordant. In the present study, red blood cell (RBC) deformability was tested via laser ektacytometry (LORCA, 0.3-30 Pa) using two approaches: 1) addition of ethanol to whole blood at 0.25%-2% followed by incubation and testing in ethanol-free LORCA medium; 2) addition of ethanol to the LORCA medium at 0.25%-6% then testing untreated native RBC in these media. The effects of ethanol on deformability for oxidatively stressed RBC were investigated as were changes of RBC aggregation (Myrenne Aggregometer) for cells in autologous plasma or 3% 70 kDa dextran. Significant dose-related increases of RBC deformability were observed at 0.25% (p < 0.05) and higher concentrations only if ethanol was in the LORCA medium; no changes occurred for cells previously incubated with ethanol then tested in ethanol-free medium. The impaired deformability of cells pre-exposed to oxidative stress was improved only if ethanol was in the LORCA medium. RBC aggregation decreased with concentration at 0.25% and higher for cells in both autologous plasma and dextran 70. Our results indicate that ethanol reversibly improves erythrocyte deformability and irreversibly decreases erythrocyte aggregation; the relevance of these results to the health benefits of moderate wine consumption require further investigation.

Analysis of laser beam scattering by an ensemble of particles modeling red blood cells in ektacytometer

Using a simple theoretical model we have obtained approximate relations between the characteristics of particles modeling red blood cells and the parameters of the diffraction pattern produced by a laser beam diffracted in the ektacytometer. Laser beam diffraction by an ensemble of transparent elliptical discs with different shapes is considered. We have shown a feasibility of measuring certain parameters of the particles distribution in shapes by using the laser ektacytometry technique.

Do We have Enough Direct Evidence to Postulate that Abnormally Shaped Red Blood Cells Impair Microvascular Blood Flow in Critical Conditions?

abnormal RBCs were produced in-vitro but not as a result of a real critical condition. The conclusions received in these two studies were confirmed in “pure” in-vivo experiments by Machiedo et al. [18]. The authors performed exchange transfusion in rats with blood obtained animals rats subjected to trauma-hemorrhagic shock or to sham shock. The reduced RBC deformability in rats that experienced traumahemorrhagic shock and normal RBC deformability in those who were subjected to sham shock was confirmed by laser ektacytometry. However, the authors did not investigate RBC shape in transfused blood. It was demonstrated that microvascular blood flow in the lungs, spleen, and intestine measured by radioactive microsphere technique was significantly lower in rats subjected to the infusion of traumahemorrhagic shock blood than in those that were infused with sham blood.

Abnormal Red Cell Deformability and Aggregation in Sickle Cell Trait

AbstractAbstract 1001 Introduction:SCT occurs in 8% of African Americans and is not commonly associated with clinical disease. Nonetheless, the United States Armed Forces has reported that SCT conveys a 30-fold risk of sudden cardiac arrest and a 200-fold risk from exertional rhabdomyolysis. In fact, rhabdomyolysis in athletes with SCT has been the principal cause of death in NCAA football players in the last decade, leading to recently mandated SCT testing in all Division-1 players. In SCT, RBC sickle only under extreme conditions and with slow kinetics. Therefore, rhabdomyolysis most likely occurs in SCT when a “perfect storm” of factors converges to critically imbalance oxygen supply and demand in muscles. We hypothesize that in SCT subjects, abnormal RBC rheology, particularly aggregation and deformability, play an important role in abnormal muscle blood flow supply and distribution to exercising muscle. To test this hypothesis, we examined whole blood viscosity, RBC aggregation, and RBC deformability in 11 SCT and 10 control subjects prior to and following maximum handgrip exercise. Methods:Maximum voluntary contraction (MVC) was assessed by handgrip dynamometer in the dominant arm. Baseline blood was collected for CBC, whole blood viscosity, RBC aggregation, and RBC deformability. Patients then maintained 60% MVC exercise until exhaustion. Following 8 minutes of recovery, a venous blood gas and blood for repeat viscosity assessments was collected from the antecubital fossa of the exercising limb. Whole blood viscosity over a shear rate range of 1–1, 000 1/s was determined by an automated tube viscometer, RBC deformability from 0.5–50 Pa via laser ektacytometry (LORCA) and RBC aggregation in both autologous plasma and 3% dextran 70 kDa using an automated cone-place aggregometer (Myrenne). Aggregation measurements included extent at stasis (M), strength of aggregation (GT min) and kinetics (T ½). Results:Baseline CBC and aggregation values are summarized in Table 1. Both static RBC aggregation in plasma and RBC aggregation in dextran (aggregability) were significantly increased in SCT (Table 1). The rate of aggregation formation trended higher in SCT but the strength of aggregation was not different between the two groups. In SCT subjects, red cell deformability was impaired at low shear stress but greater than controls at higher shear stress (Figure 1). Red cell deformability was completely independent of oxygenation status states in both SCT and control subjects. Whole blood viscosity did not different between the two groups whether oxygenated or deoxygenated and prior to or following handgrip exercise.Table 1SCTControlsP valueHemoglobin12.5 ± 0.712.3 ± 0.90.97Hematocrit36.4 ± 1.837.4 ± 2.40.08RBC4.33 ± 0.244.47 ± 0.550.32MCV84.9 ± 2.884.9 ± 8.10.30MCH28.9 ± 1.028.0 ± 3.10.83MCHC34.4 ± 0.632.9 ± 1.10.0002WBC4.65 ± 0.945.22 ± 1.610.30M plasma17.1 ± 3.812.2 ± 4.20.01M dextran23.8 ± 3.118.6 ± 6.10.02T ½ plasma2.9 ± 1.33.9 ± 1.50.08GT min plasma53.4 ± 28.450.6 ± 28.60.65GT min dextran36.5 ± 9.631.2 ± 7.90.14 [Display omitted] Discussion:Three important hemorheological differences were observed for SCT subjects versus controls: a) RBC deformability was below control at low stress levels yet greater than control at higher stress; b) The extent of RBC aggregation in autologous plasma was about 40% greater; c) The extent of RBC aggregation for washed RBC re-suspended in an aggregating medium (i.e., 3% dextran 70 kDa) was about 30% higher. RBC deformability is a major determinant of in vivo blood flow dynamics, especially in the microcirculation; decreased deformability adversely affects tissue perfusion. RBC aggregation is also an important determinant since it affects both resistance to blood flow and RBC distribution in a vascular bed (e.g., plasma skimming). The finding of greater aggregability (i.e., higher aggregation in the defined dextran medium) indicates that RBC in SCT have an altered membrane surface in which the penetration of this polymer into the glycocalyx is abnormal. The combined effects of these three rheological parameters is likely to impair in vivo blood flow in SCT, perhaps to a degree resulting in pathophysiological changes of the cardiovascular system. Disclosures:Coates:Novartis: Speakers Bureau; Apopharma: Consultancy. Wood:Ferrokin Biosciences: Consultancy; Shire: Consultancy; Apotex: Consultancy, Honoraria; Novartis: Honoraria, Research Funding.

Red blood cell damage after trauma-hemorrhage is modulated by gender.

Previous studies have shown that trauma-hemorrhagic shock (T/HS) causes significant alterations in red blood cell (RBC) deformability and shape. Gender is becoming well recognized as a modulating factor in the pathophysiologic response to trauma. We hypothesize that female subjects are more resistant to adverse effects of T/HS on RBC deformability and shape than male subjects because of estrogen protection. Elongation index, a measure of RBC deformability, was examined in six male rats and four groups of female rats at different stages of the estrous cycle (six animals per group) before and 6 hours after T/HS by laser ektacytometry. RBC shape was determined by scanning electron microscopy. Lipid peroxidation was evaluated by measurement of malonyldialdehyde in plasma 6 hours after T/HS. Male rats had a significant decrease in RBC elongation index after T/HS as compared with preshock values. RBC deformability of the proestrous (PES) and estrous female rats did not decrease after T/HS. Postshock RBC deformability values in metestrous and diestrous female rats did not differ from male rats. Male rats and metestrous and diestrous females had a significant increase in the percentage of abnormally shaped RBCs immediately after shock and during the postresuscitation period. In PES and ES female rats, RBC shape distribution did not significantly change after T/HS. Plasma malonyldialdehyde levels at 6 hours post-T/HS were higher in the male rats than in PES female rats. These data show that high levels of estrogen in female rats are protective against shock-induced RBC damage and suggest that this effect is associated with a decrease in lipid peroxidation.

Burn-induced red blood cell deformability and shape changes are modulated by sex hormones

Background Burns are known to cause changes in red blood cell (RBC) deformability and resting shape. However, it is unclear whether sex and sex hormones can influence the severity of these alterations. Methods Red blood cell deformability and shape were examined in proestrus and diestrus female rats, ovariectomized female rats, as well as castrated and noncastrated male rats (6 animals per group) subjected to scald burn. Red blood cell deformability was measured by laser ektacytometry and erythrocyte shape was evaluated by scanning electron microscopy. Results Burn-induced RBC deformability changes (decrease in elongation index) and shape alterations (increase in the percentage of reversibly and irreversibly changed cells) were less severe in proestrus females than in diestrus females or males. Ovariectomized rats demonstrated more severe RBC changes than nonovariectomized ones. The degree of RBC damage was the same in castrated and noncastrated males. Conclusions Removal of female sex hormones increases the severity of burn-induced RBC, indicating that female sex hormones protect against burn-induced RBC dysfunction. In contrast, male sex hormones do not appear to modulate burn-induced RBC dysfunction.