Evaluación de alteraciones hemorreológicas en pacientes hipertensos / Hemorheological alterations in hypertensive patients

El objetivo de este trabajo fue investigar el perfil de viscosidad sanguínea y evaluar la influencia de factores plasmáticos ( fibrinógeno) y celulares ( agregación eritrocitaria ) en un grupo de pacientes hipertensos comparados con un grupo de paciente normotensos. Se trabajó con sangre anticoagulada de pacientes hipertensos no diabéticos (n=3) e indivíduos sanos (n=40). La viscosidad plasmática y de sangre entera se determinaron con un viscosímetro cono-plato. La agregación eritrocitaria se estudió por observación microscopia de los agregados y cuantificación a través de un parâmetro de forma denominado ASP ( Aggregation Shape Parameter), definido como la relación de área proyectada respecto al perímetro. El fibrinógeno se determino con un coagulómetro por el método de Clauss. Los valores de viscosidad de sangre entera resultaron significativamente aumentados en los pacientes hipertensos respecto de los normales para todas las velicidades estudiadas. Los valores de viscosidad plasmática solo presentaron diferencia significativas a bajas velocidades de corte (1.15 a 11.56 seg •1) . Los pacientes hipertensos presentaron agregados amorfos e irregulares, lo que se refleja en los valores alterados de ASP, significativamente mayores (p<0.001) en paciente hipertensos (0.69± 0.11) respecto de los indivíduos normales ( 0.25± 0.12). Los valores de fibrinógeno resultaron ligeramente superiores en los pacientes hipertensos respecto del grupo control (p< 0.01). Numerosos parámetros hemorreológicos juegan un papel importante en la patogénesis de la hipertensión. Entre estos factores hemorreológicos, valores parâmetros podrían estar en la hipertensión ( hematrocito, fibrinógeno plasmático, deformabilidad y agragación eritrocitaria , viscosidad sanguínea y plasmática). En este trabajo, se pudo demostrar anormalidades en la agregación eritrocitaria, detectada por los valores de ASP que podría estar involucrado en las complicaciones vasculares de la hipertensión.

Evaluación de alteraciones hemorreológicas en pacientes hipertensos / Hemorheological alterations in hypertensive patients

El objetivo de este trabajo fue investigar el perfil de viscosidad sanguínea y evaluar la influencia de factores plasmáticos ( fibrinógeno) y celulares ( agregación eritrocitaria ) en un grupo de pacientes hipertensos comparados con un grupo de paciente normotensos. Se trabajó con sangre anticoagulada de pacientes hipertensos no diabéticos (n=3) e indivíduos sanos (n=40). La viscosidad plasmática y de sangre entera se determinaron con un viscosímetro cono-plato. La agregación eritrocitaria se estudió por observación microscopia de los agregados y cuantificación a través de un parÔmetro de forma denominado ASP ( Aggregation Shape Parameter), definido como la relación de área proyectada respecto al perímetro. El fibrinógeno se determino con un coagulómetro por el método de Clauss. Los valores de viscosidad de sangre entera resultaron significativamente aumentados en los pacientes hipertensos respecto de los normales para todas las velicidades estudiadas. Los valores de viscosidad plasmática solo presentaron diferencia significativas a bajas velocidades de corte (1.15 a 11.56 seg ò1) . Los pacientes hipertensos presentaron agregados amorfos e irregulares, lo que se refleja en los valores alterados de ASP, significativamente mayores (p<0.001) en paciente hipertensos (0.69± 0.11) respecto de los indivíduos normales ( 0.25± 0.12). Los valores de fibrinógeno resultaron ligeramente superiores en los pacientes hipertensos respecto del grupo control (p< 0.01). Numerosos parámetros hemorreológicos juegan un papel importante en la patogénesis de la hipertensión. Entre estos factores hemorreológicos, valores parÔmetros podrían estar en la hipertensión ( hematrocito, fibrinógeno plasmático, deformabilidad y agragación eritrocitaria , viscosidad sanguínea y plasmática). En este trabajo, se pudo demostrar anormalidades en la agregación eritrocitaria, detectada por los valores de ASP que podría estar involucrado en las complicaciones vasculares de la hipertensión. (AU)

Characterization of monoclonal antibodies against human red blood group cells antigens by laser backscattering.

A problem in immunohematology is to define the antibody quality which is related to its affinity expressed by the equilibrium constant. The activity of an antibody can be measured by the strength of its interaction, related to the adhesive energy exchanged during RBC agglutination which depends on the antigen-antibody liaison strength. To estimate this adhesive energy, two methods are used in this paper. Firstly, the dissociation behaviour of suspended RBC agglutinates was analysed by laser backscattering intensity (r) in a Couette flow. Backscattered intensity issued from shear-induced mechanical dissociation is recorded and submitted to a numerical process to obtain the energy parameter (ED). Secondly, a modification of this technique is proposed for measuring specific binding energy. Samples were exposed to increasing shear stress, and backscattered intensity was recorded. A constant increase of this intensity with raising shear stress was observed, pointed to a progressive dissociation of RBC agglutinates into smaller ones. Considering that complete dissociation of agglutinates is only approached asymptotically it is assumed that the final break-up of doublets (two-cell agglutinates) is produced at a critical shear stress (tauC) reflecting the work done to breaking-up the molecular bridges between both adjacent cells. This shear stress is defined by the extrapolation of the linear part of the curves [r-log tau] to the backscattered signal (r0) corresponding to the complete dispersion of RBCs. These approaches permit to define the specific surface adhesive energy (Gamma) by using the Derjaguin relation and to assess the functional characterization of specific immunoglobulins. In conclusion, two parameters characterizing monoclonal antibody agglutination properties, ED and Gamma, were estimated by laser backscattering methods, which could be very useful for antibodies quality control.

Evaluation of the energy of red blood cell agglutination by monoclonal antibodies.

The knowledge of the energy involved in cell-cell interactions has significant implications in biological and medical sciences. Red blood cells (RBC) become mutually adhered when specific (agglutinins) or nonspecific macromolecules bind adjacent cells in an irreversible or reversible form. Flow chamber technique with digital image processing was successfully applied to determine the partial separation, by shear stress, of two RBC agglutinated face to face (doublet) by specific monoclonal antibodies (MAb) and also to evaluate the shear stress required to attain this separation. The work done in separating adhered cells is then easily calculated and serves to evaluate the antibody affinity. In this work, this technique was applied to evaluate the affinity of a monoclonal antibody specific to the blood group A antigen. The specific disaggregation energy gamma (i.e., the work done by the shear stress) required to disrupt a unit of adhered membrane areas between agglutinated cells was calculated. On play back of the image analysis, measurements were taken to determine the force applied to the doublet and the relative separation between both RBC. Values of gamma(d) (from 2.70 to 4.61 x 10(-9) N/cm) were found to be proportional to the density (D = 27 to 55 molecules/microm(2)) of MAb molecules bound on the RBC membrane.

Correlated random walk: a fractal approach to erythrocyte viscoelastic properties.

A numerical method is proposed to evaluate the fractal correlation coefficient on viscoelastic properties of mammalian erythrocyte membranes from the diffractometric data obtained with the erythrodeformeter [16]. The numerical method is formulated on the basis of the fractal approximation for ordinary Brownian motion (OBM) and fractionary Brownian motion (FBM) [10]. Photometric readings performed on the elliptical diffraction pattern, generated by the shear elongated cells and photometrically recorded curves of creep and recovery of cells, are used in the calculations of self-affine Brownian correlation coefficient, averaged over several millions of cells. The time dependence of the correlation coefficient from different hematological disorders and also from healthy donors was calculated, and significative differences were found between both results. Diffractometric data belonging to healthy donors behaves as white noise, while data series from different disease were found to be chaotic.

Senescent erythrocytes: modification of rheologic properties, antigenic expression and interaction with monocytes.

Human erythrocytes have a well-defined lifespan of 120 days. Their eventual removal from circulation is a complex process affected by many cellular parameters, making them susceptible to sequestration in the spleen and other organs. The purpose of this study was to investigate putative changes in rheologic properties, antigenic expression and interaction with monocytes of senescent erythrocytes (SE). SE and young erythrocyte (YE) fractions were obtained by differential centrifugation from 20 healthy donor blood samples. Membrane rheomechanic properties (by diffractometric method), ABO and MN antigens reactivity and erythrophagocytosis by peripheral monocytes were investigated in each fractions. SE showed a little decrease in the deformability index and an increase of both membrane elastic modulus and surface viscosity. The studies performed indicate a decreased expression in the antigens of both blood group systems studied (p < 0.01) and an increased rate of erythrophagocytosis by monocytes in SE compared to YE (p < 0.01). The significant modifications in the biomechanic properties of senescent red blood cell membrane and the loss of antigenic expression could lead to physiological phagocytosis.

Senescent erythrocytes: modification of rheologic properties, antigenic expression and interaction with monocytes.

Human erythrocytes have a well-defined lifespan of 120 days. Their eventual removal from circulation is a complex process affected by many cellular parameters, making them susceptible to sequestration in the spleen and other organs. The purpose of this study was to investigate putative changes in rheologic properties, antigenic expression and interaction with monocytes of senescent erythrocytes (SE). SE and young erythrocyte (YE) fractions were obtained by differential centrifugation from 20 healthy donor blood samples. Membrane rheomechanic properties (by diffractometric method), ABO and MN antigens reactivity and erythrophagocytosis by peripheral monocytes were investigated in each fractions. SE showed a little decrease in the deformability index and an increase of both membrane elastic modulus and surface viscosity. The studies performed indicate a decreased expression in the antigens of both blood group systems studied (p < 0.01) and an increased rate of erythrophagocytosis by monocytes in SE compared to YE (p < 0.01). The significant modifications in the biomechanic properties of senescent red blood cell membrane and the loss of antigenic expression could lead to physiological phagocytosis.

[Blood preservation. Bacteriological, immunohematological, hematological and hemorrheological studies]. / Almacenamiento de sangre. Estudios hemorreológicos, hematológicos, inmunohematológicos y bacteriológicos.

The knowledge of erythrocyte viability during blood bank storage period has been a major problem for transfusional practice. This problem must be considered by taking into account blood flowing properties which are directly related to the rheological properties of erythrocytes. The aim of this work was to perform an interdisciplinary study of the modifications induced in erythrocyte properties during 29 days storage of whole blood in a blood bank. Three samples of venous blood collected from healthy adults in plastic bags, anticoagulated with ACD (citric acid, citrate, dextrose) and stored at 4 degrees C were studied. Rheological properties (deformability, membrane elasticity and surface viscosity), immunohematological conditions (antigenicity) of erythrocytes, haematological index variation and bacteriological cultures of blood were weekly analysed. Rheological studies were performed using the Erythrodeformeter, a polymicroviscosimeter (paper filter) and erythrocyte filtration through polycarbonate membranes of 5 microns pore diameter. Impairment of membrane rheological properties, loss of antigenicity and decreases of Ht, Hb, MCHC and MHC were observed. Bacteriological cultures gave always negative results. The studies performed showed significant alterations of erythrocyte rheological parameters. It becomes evident that such modifications would influence the erythrocyte mechanical behavior in microcirculation and impair the in vivo viability of the transfused red cells.

Complex viscoelasticity of normal and lectin treated erythrocytes using laser diffractometry.

A new method to find directly complex viscoelastic parameters (CVP) of red blood cells (RBC) is presented in this paper. Experimental determinations were carried out in an Erythrodeformeter (Rasia et al., 1986) 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. The measurements lead to the determination of mean values of the four components of erythrocyte complex viscoelasticity. The influence of the alterations induced on erythrocyte membrane by vegetable lactins (Ulex europaeus, wheat germ agglutinin and Enterolobium contorticilicum seeds) was analyzed to verify the sensitivity of this method. Differences observed between the CVP parameters of treated cells and the ones corresponding to control samples (non treated cells) are analyzed. Results obtained from cells treated with wheat germ agglutinin agree with observations published by Smith and Hochmuth (1982). Determinations of RBC complex viscoelasticity carried out by laser diffractometry could become an important tool to understand the influence of the factors associated with alterations of the rheologic properties of RBC membrane, which can affect the in vivo blood flow.

Rheologic characterization of vegetal lectins by dissociation of induced erythrocyte agglutinates.

Energy evolved from hemagglutination reaction or spent in dissociating erythrocyte agglutinates has been proved to be an excellent parameter for analyzing cell-cell interactions mediated by bridging molecules such as antibodies or lectins. We developed a new rheo-optical method to estimate the energy of dissociation of red blood cell agglutinates. In a Couette shear field agglutinates can be dissociated until a suspension of monodispersed cells is obtained. Intensity of light backscattered by suspended agglutinates increases during their mechanical dissociation. Variation of backscattered light intensity correlates with the energy spent in the process. The adhesive energy of erythrocyte agglutination induced by lectins has been estimated by applying this method. Two specific lectins (Dolichus Biflorus agglutinin and Ulex Europaeus agglutinin) and a new lectin obtained from Amarantus Cruentus seeds which specificity is unknown were studied. Results obtained in this work for Dolichus Biflorus lectin are comparable with values published by other authors. An asymptotic decrease of adhesive energy was observed when the mechanical dissociation was applied several times on the same sample. Our results suggest that the cell detachment is accompanied by the extraction of membrane receptors. This finding is consistent with results obtained by other authors.

[Rheologic study of the dissociation reaction of erythrocyte immunologic agglutinates: application to subgroup A erythrocytes]. / Approche rhéologique de la réaction de dissociation d'agglutinats immunologiques érythrocytaires: application à des hématies du sous-groupe A.

Red blood cell Agglutinates can be dissociated by providing enough energy which may be supplied by shear stress in a viscometer. In this paper a new technique is proposed to determine dissociation parameters of erythrocyte immunological agglutinates by using a laser backscattering method. Backscattered light intensity increases during the process of agglutinate dissociation when a controlled shear stress in a transparent Couette viscometer is applied. The obtained curves of dissociation can be fitted by an exponential function. A numerical integration of the dissociation curves allows us to obtain a parameter associated to the energy used in the dissociation process. A satisfactory differentiation of erythrocyte sub-groups Al, Az, A,B and Am has been carried out using this technique.

[Effect of cholesterol on the cellular deformability and osmotic fragility of erythrocytes]. / Influence du cholestérol sur la déformabilité cellularire et la fragilité osmotique des hématies.

The effect of cholesterol hemisuccinate incorporation on the rheological properties of the erythrocyte membrane was assessed by measuring deformability (elongation index) and osmotic fragility ("baseline hemolysis"). These two experimental methods have different response sensitivities according to the incubation protocols (12 h 25 degrees C/12 h 25 degrees C then 12 h 37 degrees C) ant to the amount of cholesterol incorporated in the membrane (C/Pt from 0.18 to 0.83). The deformability and osmotic fragility variations observed are inversely related with the cholesterol concentrations incorporated. The experimental differences observed in the deformability and osmotic fragility are discussed in view of the type of membrane deformation and of the signals collected by the two systems.

Manual quantitative method for the study of red cell agglutination using light diffraction by suspended particles.

A new manual technique, called the diffractometric technique, for quantitation of hemagglutination is described in this paper. After the equilibrium has been attained in the reaction, the whole product is resuspended in a dense transparent medium in order to obtain a stable suspension. The relative optical extinction produced by this suspension depends on the number and sizes of the suspended particles and on the photodetector geometry. Hence the agglutination percentage of the reaction can be monitored by using a visible light transmittance photometer. The results of this technique compare very well with the ones obtained by applying two other methods, the Dybkjaer and the Ropars methods.

[Evaluation of the kinetics of hemagglutination using a simple photometric technic]. / Evaluación de la cinética de hemaglutinación por medio de una técnica fotométrica simple.

A photometric technique to evaluate haemagglutination kinetic is presented in this paper. The technique applies the relative optical extinction (EO) produced on a light beam transmitted through a suspension of small particles (red blood cells and their agglutinates). The optical extinction decreases as the red cell agglutinates grow, giving a parametric estimate of the haemagglutination rate. Hence, optical extinction can be used to distinguish erythrocyte subgroups characterized by the differences appearing in the number of antigenic sites per cell. The paper describes the technique and gives the results of a set of experiments carried out on 90 samples constituted by 30 samples of A1 adults, 30 samples of non-A1 A adults and 30 samples of A newborns (cord red blood cells). Results show significative differences between subgroups. Parametric values such as the total relative optical extinction reduction (difference between the initial and the final values of EO) and the initial optical extinction rate are analyzed. Results from A newborn red cells show similar behaviour as the non-A1 A adult red cells and significative differences from A1 adult red cells. A correlation between parametric values and the number of antigenic sites per cell, as found in the literature, becomes evident.