We analyze the erythrocyte aggregation by the biospeckle laser phenomenon and calculate characteristic parameters to develop non-invasive hemorheological techniques. Biospeckle is more complex than other light-based techniques, such as light transmission, providing information about the dynamics of the scatterers, their size, and morphology. Dextran 500 kDa combined with plasma and phosphate-buffered saline was used to induce controlled in vitro erythrocyte aggregation. Red blood cell aggregation was studied using an objective speckle setup in a microchip sample chamber. The speckle grain sizes were determined, and parameters to characterize the morphology of the erythrocyte aggregates were proposed. The inertia moment and the correlation coefficient were determined to assess the cells aggregation dynamics.
In this work, we studied the nature of the molecular bonds involved in the red blood cell aggregation process by the coherent anti-Stokes Raman spectroscopy technique. Images were acquired with a commercial Leica TCS SP8 CARS confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) temporally and spatially overlapping the pulses of two sources in the focal plane of the microscope. A pump wavelength of 810 nm to 817 nm was used for the CARS mode simultaneously with the Stokes beam at 1064 nm to excite the vibratory resonance of the symmetric hydrocarbon bonds in the lipids and that of the bonds in amino acids of the proteins. The Raman shift was also observed at the 1200 cm−1 range to study possible variations in the sialic acid on the cell membrane produced by concentrations of dextran 500 in the suspension medium. Curves of lifetime emission distribution were obtained for untreated erythrocytes and treated erythrocytes with dextran 500, particularly at a pump wavelength of 904 nm.
The study of erythrocyte (RBC) aggregation is of great interest because of its implications on human health, alterations in erythrocyte aggregation can lead to microcirculatory problems. An optical-chip based system was developed using laser transmission techniques in order to evaluate and characterize RBC aggregation. Studies are carried out with in vitro altered RBC by non-enzymatic glycosylation. Several samples were analyzed, and by computational data processing, characteristic parameters were found, describing RBC aggregation kinetics in order to improve the early detection in clinical environments of these anomalies, generally present in vascular diseases such as hypertension and diabetes.
The study of red blood cell (RBC) aggregation is of great interest because of its implications for human health. Altered RBC aggregation can lead to microcirculatory problems as in vascular pathologies, such as hypertension and diabetes, due to a decrease in the erythrocyte surface electric charge and an increase in the ligands present in plasma. The process of erythrocyte aggregation was studied in stasis situation (free shear stresses), using an optical chip based on the laser transmission technique. Kinetic curves of erythrocyte aggregation under different conditions were obtained, allowing evaluation and characterization of this process. Two main characteristics of blood that influence erythrocyte aggregation were analyzed: the erythrocyte surface anionic charge (EAC) after digestion with the enzyme trypsin and plasmatic protein concentration in suspension medium using plasma dissolutions in physiological saline with human albumin. A theoretical approach was evaluated to obtain aggregation and disaggregation ratios by syllectograms data fitting. Sensible parameters (Amp100, t1\2) regarding a reduced erythrocyte EAC were determined, and other parameters (AI, M-Index) resulted that are representative of a variation in the plasmatic protein content of the suspension medium. These results are very useful for further applications in biomedicine.
Red blood cell aggregation is one of the most important factors in blood viscosity at stasis or at very low rates of flow. The basic structure of aggregates is a linear array of cell commonly termed as rouleaux. Enhanced or abnormal aggregation is seen in clinical conditions, such as diabetes and hypertension, producing alterations in the microcirculation, some of which can be analyzed through the characterization of aggregated cells. Frequently, image processing and analysis for the characterization of RBC aggregation were done manually or semi-automatically using interactive tools. We propose a system that processes images of RBC aggregation and automatically obtains the characterization and quantification of the different types of RBC aggregates. Present technique could be interesting to perform the adaptation as a routine used in hemorheological and Clinical Biochemistry Laboratories because this automatic method is rapid, efficient and economical, and at the same time independent of the user performing the analysis (repeatability of the analysis).
Traditional techniques to evaluate the aggregation of red blood cells by optical methods require large sample volume and provide parameters that vary significantly from one method to another. A simplified variant of a chip system previously developed by Shin et al. (2009)1 based on light transmission for measuring erythrocyte aggregation is presented. Through a detailed analysis of intensity versus time curves, relevant information about erythrocyte aggregation and its variables is obtained. Parameters that provide more accuracy for the diagnosis of patients in order to have an immediate application in Clinical Medicine are proposed.
KEYWORDS: In vitro testing, Image analysis, Blood, Simulation of CCA and DLA aggregates, Statistical analysis, Surgery, Digital breast tomosynthesis, Digital cameras, Plasma, Vascular diseases
Drugs used during anesthesia could enhance microvascular flow disturbance, not only for their systemic cardiovascular actions but also by a direct effect on the microcirculation and in particular on hemorheology. This is particularly important in high-risk surgical patients such as those with vascular disease (diabetes, hypertension, etc.). Therefore, in this work we propose a set of innovative parameters obtained by digital analysis of microscopic images to study the in vitro hemorheological effect of propofol and vecuronium on red blood cell from type 2 diabetic patients compared to healthy donors. Obtained innovative parameters allow quantifying alterations in erythrocyte aggregation, which can increase the in vivo risk of microcapillary obstruction.
The aim of this work was to study synthetic polycation effects on erythrocyte agglutination mediated by anti-glycophorin
using image digital analysis. Polycations are oligomers or polymers of natural or synthetic origin, which bear a great
number of positive charges at pH 7.4. Several of these polycations are nowadays used in clinic for human and veterinary
purposes. New applications of polycations to the development of new drug delivery systems are investigated, in order to
promote the drug absorption through the gastro-intestinal and blood brain barriers. However, up to now, there are no
clear relationships between macromolecular features of polycations (molecular weight, mean charge density, charge
repartition, etc.) and their interactions with blood elements (which bear superficial negative charges). The interaction on
the red blood cell membrane with synthetic polycations having well-controlled macromolecular features and
functionalized with pendent polyethylene glycol segments was investigated. The alterations over stationary and dynamic
viscoelastic properties of erythrocyte membranes were analyzed through laser diffractometry. Image digital analysis was
used to study erythrocyte agglutination mediated by anti-glycophorin. Results show different reactivities of the
polycations on the erythrocyte membrane. These findings could provide more information about the mechanisms of
polycation interaction on erythrocyte membranes. We consider that this work could provide useful tools to understand
and improve the haemocompatibility of polycations and enlarge their potential in clinic.
We report the adhesion of human erythrocyte membranes mediated by monoclonal antibodies anti-glycophorin. The distribution of the linked antibodies on membrane was identified with selective fluorescence labels. To analyze the antibody distribution on interfacial region between two cells agglutinated and on its surface, three types of fluorescence marked strategy were evaluated. The 3D images were obtained in a CellScan and Confocal Laser Scanning Microscopy CLSM. We considered the FRET signal to characterize the agglutination of Red Blood Cells (RBC) by specific monoclonal antibodies (anti-glycophorin A or B). The fluorescence labeling demonstrated that distribution of antibody on erythrocyte membranes is not homogeneous. The fluorescence intensity on contact region in the agglutinated is bigger than the intensity on exterior surface. Tentatively, we interpreted these intensity differences in terms of the mobility of antibody linked to the glycocalix on cell surface. Such mobility has a large consequence in the morphology of cellular agglutinated.
In this work we investigated the erythrocyte membrane viscoelastic behavior, in hypertensive and dislipidemic patients using the "Erythrodeformeter" that permit to obtain the stationary and dynamical linear parameters of erythrocyte membrane by laser diffractometry. Our results show that several erythrocyte membrane rheological parameters were statistically altered in patients if compared with the control group. Then, the analyzed hemorheological parameters could be use in order to detect and diagnostic the hypertension and dyslipidemic alterations.
Detection of antigen is carried out by means of an optical immunosensor that uses laser reflectometry technique. It consists in a silicon wafer with antibody adhered on surface. Measurements were performed in samples containing different concentration of antigen A. This technique is very sensitive, quick and independent of cellular properties.
A new method to find directly complex viscoelastic parameters (CVP) of human red blood cells is presented in this paper. Experimental determinations were carried out in an Erythrodeformeter operating in oscillating mode (0.5 to 3.5 Hz). The Erythrodeformeter performs direct determination of CVP of erythrocytes undergoing sinusoidal shear stresses by laser diffractometry.
Study of biomolecular interaction dynamics and relations between their structure and function is very important for the understanding of biological system. The laser diffractometry technique, as well as the surface plasmon resonance technique, can give information about the interaction kinetics, molecular binding stoichiometry, and the concentration of molecules in a solution. This method offers detailed and accurate determination of real-time adsorption kinetics of protein without labeling of the protein. When the polarized light is reflected on a solid surface, the reflectance has a minimum at the principal incidence angle. As the proteins are adsorbed over the surface the reflectance increases. Therefore, the temporal register of reflected intensity gives curves representing the binding kinetics of antibody in real-time. The technique is highly sensible because of the great difference in the refractive index between silicon and organic material. This method was applied to study the adhesion kinetics of monoclonal anti-AB antibodies at surface of a silicon wafer. The mathematical analysis of results would suggest that the antibody association occurs according to classical Langmuir surface adsorption. This analysis permits to obtain the adhesion and dissociation of the antibody.
An optical method has been developed to find (alpha) 1- antitrypsin unknown concentrations in human serum samples. This method applies light scattering properties exhibited by initially formed enzyme-inhibitor complexes and uses the curves of aggregation kinetics. It is independent of molecular hydrodynamics. Theoretical approaches showed that scattering properties of transient complexes obey the Rayleigh-Debie conditions. Experiments were performed on the Trypsin/(alpha) 1-antitrypsin system. Measurements were performed in newborn, adult and pregnant sera containing (alpha) 1-antitrypsin in the Trypsin excess region. The solution was excite by a He-Ne laser beam. SO, the particles formed during the reaction are scattering centers for the interacting light. The intensity of the scattered light at 90 degrees from incident beam depends on the nature of those scattering centers. Th rate of increase in scattered intensity depends on the variation in size and shape of the scatterers, being independent of its original size. Peak values of the first derivative linearly correlate with the concentration of (alpha) 1-antitrypsin originally present in the sample. Results are displayed 5 minutes after the initiation of the experimental process. Such speed is of great importance in the immuno-biochemistry determinations.
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