Methods: A new protocol for in vitro red blood cell glycation.

During diabetes, the characteristic hyperglycemia can induce red blood cell glycation. Several researchers have proposed different protocols to perform an in vitro model to study this phenomenon. In this article, some of the most important in vitro glycation protocols available in the bibliography were compared to each other. The incubation parameters as the suspension medium, glucose concentration, red blood cell concentration, time, and temperature were analyzed. Also, several assays were carried out in our laboratory, and glycated hemoglobin, erythrocyte aggregation and viscoelasticity were determined for the protocol validation. Based on the bibliographic analysis and our experimental results, an optimal protocol for in vitro glycation of red blood cells is presented.

In vitro alteration on erythrocytes mechanical properties by propofol, remifentanil and vecuronium.

Several studies report flow disturbance and microcirculation disorders upon anesthesia treatment. These alterations are often related to blood rheology changes. In this work, it was attempted to make a detailed description of the alterations in erythrocyte mechanical properties by the action of propofol, remifentanil, and vecuronium. For this, an in vitro study was performed on red blood cell samples from healthy donors incubated with solutions of propofol (4 µg/mL whole blood), remifentanil (10 ng/mL plasma), and vecuronium (0.15 µg/mL plasma). Erythrocyte viscoelastic parameters were determined by octuplicate using a Reómetro Eritrocitario. Also, a Wilcoxon signed rank-test with Yates correction for continuity was performed to analyze the overall alteration in the mechanical properties of erythrocytes. Statistical analysis showed that the three studied anesthetics changed the erythrocyte mechanical properties at different parts of the membrane. These results would imply an interaction of these anesthetics with the erythrocyte membrane. Finally, this could conduce to alterations in microcirculation.

Simultaneous Determination of Human Erythrocyte Deformability and Adhesion Energy: A Novel Approach Using a Microfluidic Chamber and the "Glass Effect".

The simultaneous determination of adhesion and deformability parameters of erythrocytes was carried out through a microfluidic device, which uses an inverted optical microscope with new image acquisition and analysis technologies. Also, an update of the models describing erythrocyte adhesion and deformation was proposed. Measurements were carried out with red blood cells suspended in saline solution with human serum albumin at different concentrations. Erythrocytes adhered to a glass surface were subjected to different low shear stress (from 0.04 to 0.25 Pa), causing cellular deformation and dissociation. The maximum value obtained of the erythrocyte deformability index was 0.3, and that of the adhesion energy per unit area was 1.1 × 10-6 Pa m, both according to previous works. The obtained images of RBCs adhered to glass reveal that the adhesion is stronger in a single point of the cell, suggesting a ligand migration that concentrates the adhesion in a "spike-like tip" in the cell. Moreover, adhesion energy results indicate that the energy required to separate erythrocytes in media with a lower albumin concentration is greater. Both results could be explained by the mobility of membrane receptors.

Caracterización de cristales líquidos con Miconazol en sistemas bifásicos por microscopía de polarización / Characterization of liquid crystals with Miconazole in biphasic systems by polarized microscopy

INTRODUCCIÓN: las emulsiones son un tipo de preparado farmacéutico muy utilizado en aplicación tópica consistentes en sistemas bifásicos de aceite-agua o agua-aceite, donde el principio activo deseado se incorpora en una de las fases dependiendo de su solubilidad. Diversos estudios demuestran que la estabilidad es mayor en emulsiones con estructuras líquido-cristalinas. El principio activo liposoluble Miconazol, que actúa como antimicótico, se solubiliza en la fase oleosa de la emulsión y en la fracción de la cadena hidrocarbonada de los cristales líquidos. MÉTODO: se utilizaron técnicas microscópicas para analizar las características de una emulsión convencional y de otra con cristales líquidos, a las que se les incorporó el principio activo Miconazol. Se determinaron las dimensiones de las gotas de la fase interna y mediante microscopía de polarización se caracterizaron los cristales líquidos. RESULTADOS: el análisis de las imágenes microscópicas permitió determinar que en las formulaciones con cristales líquidos con y sin Miconazol, aproximadamente el 80 % de las gotas tienen dimensiones en el intervalo 0,5 mim - 1 mim. Las observaciones microscópicas con luz polarizada nos permitieron determinar que los cristales líquidos tienen birrefringencia con la formación de cruces de extinción uniáxicas negativas, las cuales son características de las fases liotrópicas laminares con texturas cónicas focales. CONCLUSIONES: los resultados muestran que el agregado de Miconazol, no interfiere con la formación de la estructura de los cristales líquidos, por lo que estas dependen de los componentes de la formulación y de la técnica de preparación

INTRODUCTION: emulsions are a type of pharmaceutical preparation widely used in topical applications consisting of two-phase systems of oil-in-water or water-in-oil, where the desired active ingredient is incorporated into one of the phases depending on its solubility. Several studies show that stability is greater in emulsions with liquid-crystalline structures. The liposoluble active substance Miconazole, which acts as an antifungal agent, is solubilized in the oil phase of the emulsion as well as in the fraction of the hydrocarbon chain in liquid crystals. METHOD: microscopic techniques were used to analyze the characteristics of both a conventional emulsion and another one containing the liquid crystals. Miconazole was incorporated into both emulsions; drop dimensions in the internal phase were determined and the liquid crystals were characterized by polarization microscopy. RESULTS: through the analysis of the microscopic images of the formulation with liquid crystals with Miconazole and without Miconazole, it was possible to determine that approximately 80% of the drops have dimensions ranging from 0.5 Mum - 1 Mum. Microscopic observations with polarized light allowed us to determine that liquid crystals have birefringence with the formation of negative uniaxial extinction crosses, which are characteristic of laminar lyotropic phases with focal conical textures. CONCLUSIONS: the results show that the addition of Miconazole does not interfere with the formation of the structure of the liquid crystals. Therefore, the formation of liquid crystals depends both on the components of the formulation and the preparation technique

Descriptive parameters of the erythrocyte aggregation phenomenon using a laser transmission optical chip.

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 ( Amp 100 , t 1 \ 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.

Hemorheological in vitro action of propofol on erythrocytes from healthy donors and diabetic patients.

Drugs used during anesthesia might induce disturbance on microcirculation due to their systemic cardiovascular actions and to direct hemorheological effects. A comparative investigation of the hemorheological alterations related to in vitro propofol treatment of red blood cells (RBCs) from healthy and diabetic volunteers is presented here. Viscoelasticity and aggregation of RBCs from type 2 diabetic patients (DBT) and healthy donors (HD) were studied from RBCs incubated with propofol near steady-state concentration. 'S parameter', which measures the aggregation degree, was obtained using digital analysis of microscopic images. Erythrocyte viscoelasticity parameters were determined using an Erythrocyte Rheometer. Results obtained from DBT samples showed an increase of 10% or more in aggregation due to the propofol action. The phase shift between erythrocyte response and oscillating shear stress applied at 1 Hz was altered by propofol treatment of erythrocyte from HD and DBT. Propofol could produce slight alterations in the rheological behavior of erythrocyte from HD and DBT, at concentrations near those of steady state. Moreover, this anesthetic could induce an adverse effect in DBT, particularly on erythrocyte aggregation. The observed hemorheologic alteration would increase the possibility of microcapillary obstruction. Hence, this type of study [0] would prove relevant to avoid possible postoperative complications.

Hemocompatibility and biofunctionality of two poly(2-(dimethylamino)ethyl methacrylate-co-poly(ethyleneglycol) copolymers.

To mask the antigenic sites of cells for cell therapies, especially for blood transfusion, we investigated the hemocompatibility of two poly(2-(dimethylamino)ethyl methacrylate-co-poly(ethyleneglycol) compared with that of the homopolymer without PEG. Our strategy relies on the potential ability of these copolymers to self-assemble at the erythrocyte surface. The cationic sequence of the copolymer should be able to interact with the glycocalyx by ionic interaction. The other sequence, based on a polyethyleneglycol moiety, should prevent both nonspecific interactions and specific recognition of the biological surface. The hemocompatibility of these copolymers was assessed by analyzing alterations in human erythrocyte membrane viscoelasticity, morphology, granularity, and aggregation. Their properties to mask ABO system and three erythrocyte glycophorin sites were investigated. No alterations in the erythrocyte morphology were observed by confocal microscopy. On the other hand, a partial masking of different specific glycophorin sites leads to future optimization of the macromolecular structures of these functionalized copolymers.

Cold-agglutinin hemolytic diseases, a rheo-optical study.

The aim of this study was to analyze the strength of red blood cells agglutination, induced by autoantibodies in patients with Cold-Agglutinin Hemolytic Disease (CAHD), and the hemorheological profile (deformability and osmotic fragility) by the utilization of rheo-optical techniques. The strength of the antigen-antibody reaction was approached by the work required to dissociate mechanically red blood cells agglutinates. It is focused on the evaluation of the qualitative adhesiveness of cell approached by the dissociation kinetics carried out in a Couette flow (erythroaggregameter). The analysis was performed by recording the increase of the reflectivity signal as the agglutinates are dissociated by shear into smaller ones. A total of eight patients aged <54 years with recent diagnostic of CAHD detected by positive Direct Anti-globulin Test (DAT) and very low RBC counts at 20 degrees C, were studied. Two parametric values were interesting: the dimensionless energy parameter and the characteristic dissociation time, which showed good correlation with hematological parameters. In conclusion, the dissociation method provides a powerful tool for estimating the qualitative adhesiveness of red blood cells agglutinated by autoantibodies in patients suffering of cold-agglutinin hemolytic disease and it would be very interesting to evaluate the severity of the disease.

A simple method for quantifying high density antigens in erythrocyte membrane by flow cytometry.

RBC flow cytometric analysis is usually used to quantify antigen content. Calibration systems enable antigen content determination by relating mean fluorescence intensity with the number of bound antibody molecules (equivalent to the number of antigen molecules). For that reason, antibodies must be used at saturating concentration, which may lead to agglutination when working with high density antigens. Then, forward scattering, side scattering and fluorescence will be increased, thus obtaining wrong results. In this work, the simple Langmuir adhesion model was applied. Flow cytometry was used to quantify GPA, a transmembrane protein present at high density on RBC. The fluorescence intensity of samples at different anti-GPA sub-saturating concentrations was measured. Sometimes, agglutinates were present and two peaks of fluorescence were observed, the principal one corresponding to isolated cells and the secondary one corresponding to agglutinated cells. In those cases, the principal peak was taken into account for the analysis. The GPA antigen content obtained for nine analyzed samples ranged from 3 to 13 x 10(5) sites per cell, which is similar to those values found in literature. Therefore, the Langmuir adsorption model enables us to determine the antigen content for the anti-GPA/GPA system on RBC membrane. This model could be used to quantify high density antigens in RBC and in other cells.

Quantification of glycophorin A and glycophorin B on normal human RBCs by flow cytometry.

BACKGROUND: The quantification of antigens and proteins on RBCs has been achieved by different approaches. Flow cytometry allows the results of the earliest studies to be to reappraised because it offers the possibility of measuring the immunofluorescence intensity of single cells and integrating the individual data of a large number of cells within a very short time. STUDY DESIGN AND METHODS: Flow cytometry was used in this work to analyze the binding of four MoAbs to glycophorin A (GPA) and glycophorin B (GPB). RBCs in their native state (nonfixed) were utilized. To avoid the agglutination problem, cells were disaggregated before measurements, dates were taken on 20,000 events on the single-cell region, and the fluorescence intensity of the principal peak present in the fluorescence histograms was used for the analysis. The quantification of sites per RBC was estimated by applying the Langmuir adhesion model. RESULTS: The numbers of GPA and GPB sites obtained for samples from healthy donors were similar to those found in the literature (1.86-4.9) x 10(5) and (0.48-1.61) x 10(5) for GPA and (0.21-1.14) x 10(5) and (0.47-0.88) x 10(5) for GPB. Differences between antibodies were found that depend on the binding site of each one. CONCLUSION: A simple method to quantify antigen sites on RBCs was developed. It could be applied whenever one antibody is assumed to bind exactly one antigen.

Viscoelasticidad compleja de los eritrocitos por difractometría láser / Complex viscoelasticity of erythrocyte by laser difractometry

El objetivo de este estudio fue investigar las propiedades reológicas de los glóbulos rojos sometidos a tensiones pulsantes (reología completa), es decir, en condiciones más próximas a las fisiológicas. La reología compleja demostró su alta sensibilidad para detectar anormalidades estructurales en cuerpos viscoelásticos, tales como sangre o eritrocitos. En este trabajo, las propiedades viscoelásticas complejas eritrocitarias fueron evaluadas con un Erotrodefórmetro, construido en este laboratorio, el cual aplica el método difractométrico en suspensiones de glóbulos rojos cizalladas. Tensiones de corte sinusoidales de frecuencia y amplitud controladas, son aplicadas a la suspensión celular. La elongación de los eritrocitos sigue las variaciones de la tensión, pero con un desfasaje angular Ù que es función de la frecuencia y de las propiedades reológicas de las células. Las variaciones de elongación se reproducen en el patrón de difracción laser y son fotométricamente medidas, digitalizadas y almacenadas para su posterior procesamiento. El procesamiento numérico de los datos experimentales conduce a la evaluación de los parámetros viscoelásticos complejos (Ù, n', n", G', G"). Los resultados obtenidos demuestran que la ektacitometría es un excelente método para analizar la reología compleja eritrocitaria y puede ser aplicado con éxito en Hemorreología Clínica para detectar anormalidades celulares patológicas

Viscoelasticidad compleja de los eritrocitos por difractometría láser / Complex viscoelasticity of erythrocyte by laser difractometry

El objetivo de este estudio fue investigar las propiedades reológicas de los glóbulos rojos sometidos a tensiones pulsantes (reología completa), es decir, en condiciones más próximas a las fisiológicas. La reología compleja demostró su alta sensibilidad para detectar anormalidades estructurales en cuerpos viscoelásticos, tales como sangre o eritrocitos. En este trabajo, las propiedades viscoelásticas complejas eritrocitarias fueron evaluadas con un Erotrodefórmetro, construido en este laboratorio, el cual aplica el método difractométrico en suspensiones de glóbulos rojos cizalladas. Tensiones de corte sinusoidales de frecuencia y amplitud controladas, son aplicadas a la suspensión celular. La elongación de los eritrocitos sigue las variaciones de la tensión, pero con un desfasaje angular U que es función de la frecuencia y de las propiedades reológicas de las células. Las variaciones de elongación se reproducen en el patrón de difracción laser y son fotométricamente medidas, digitalizadas y almacenadas para su posterior procesamiento. El procesamiento numérico de los datos experimentales conduce a la evaluación de los parámetros viscoelásticos complejos (U, n, n", G, G"). Los resultados obtenidos demuestran que la ektacitometría es un excelente método para analizar la reología compleja eritrocitaria y puede ser aplicado con éxito en Hemorreología Clínica para detectar anormalidades celulares patológicas