A caracterizagáo de agregados eritrocitários é importante para analisar as possíveis alteragóes na microcirculagáo observadas em certas patologias vasculares como a hipertensáo e a diabetes. O objetivo deste trabalho foi padronizar uma técnica que possa ser utilizada por qualquer operador com um equipamento similar. Para isso foram preparadas diversas suspensóes de glóbulos vermelhos de doadores saudáveis em plasma autólogo, que foram observadas com um microscópio óptico invertido. Foram registradas para a sua análise as imagens dos agregados com uma camera digital. Realizaramse as recontagens de células individuais, agregados de 2 a 4 células, agregados de 5 ou mais células e amas (redes de agregados de grande tamanho). Foi medido o perímetro e a área, obtendo um parametro de forma (ASP) de cada agregado de 5 ou mais células. Os resultados obtidos permitiram padronizar o protocolo de trabalho concluindo que a diluigáo ótima de glóbulos vermelhos em plasma autólogo para esta técnica é de 0,5%.

Characterization of erythrocyte aggregates is important in the analysis of the possible alterations observed in the microcirculation of certain vascular pathologies such as hypertension and diabetes. The objective of this work was to standardize a technique that can be used by any operator having similar equipment. For that purpose, different suspensions of red blood cells from healthy donors were prepared in autologous plasma and then observed with an inverted light microscope. The images of the aggregates were recorded with a digital camera in order to be later analyzed. Individual cell count was carried out, as well as 2 to 4 cell- aggregates, 5 or more cell- aggregate and amas (big aggregate networks). Measurement of perimeter and area of each of the aggregates made up of 5 or more cells was performed, getting a shape parameter (ASP). Due to the results obtained, this working protocol has been standardized and it can be concluded that the optimal dilution of red blood cells in autologous plasma is 0.5% for this particular technique.

La caracterización de agregados eritrocitarios es importante para analizar las posibles alteraciones en la microcirculación observadas en ciertas patologías vasculares como la hipertensión y la diabetes. El objetivo de este trabajo fue estandarizar una técnica que pueda ser utilizada por cualquier operador con un equipamiento similar. Para ello se prepararon distintas suspensiones de glóbulos rojos de dadores sanos en plasma autólogo, que fueron observadas con un microscopio óptico invertido. Se registraron para su análisis las imágenes de los agregados con una cámara digital. Se realizaron los recuentos de células individuales, agregados de 2 a 4 células, agregados de 5 ó más células y amas (redes de agregados de gran tamaño). Se midió el perímetro y el área, obteniéndose un parámetro de forma (ASP) de cada agregado de 5 ó más células. Los resultados obtenidos permitieron estandarizar el protocolo de trabajo concluyendo que la dilución óptima de glóbulos rojos en plasma autólogo para esta técnica es 0,5%.

Polycation - polyethylenglycol copolymers (Semo B86 and Semo B124) were developed for therapeutic uses, especially for the masking of antigenic sites of human erythrocytes to decrease the risk of aloimmunization in blood transfusions. The aim of this work was to optimize the protocol for treating human erythrocytes using these two copolymers in order to improve their hemocompatibility. The influence of the incubation time of human erythrocytes with both copolymers and the alterations produced to the morphology and erythrocyte aggregation capacity were studied using microscopy techniques and digital image analysis. Erythrocytes treated with both copolymers showed no changes in their cellular morphology but alterations in the size distribution and structure of aggregates, which were revealed by an increase in the individual cell percentage and a decrease in the percentage of large aggregates. These effects became more marked as the incubation time increased and were greater for Semo B86. These results enable a better understanding of membrane-polycation interactions and the optimization of the protocol to treat erythrocytes using these compounds. This finding may also be applied to the study of copolymers of similar structure and different therapeutic uses.

Los copolímeros policatión-polietilenglicol Semo B86 y Semo B124 fueron desarrollados con fines terapéuticos, en particular, el enmascaramiento de sitios antigénicos de eritrocitos humanos para disminuir el riesgo de aloinmunización en transfusiones sanguíneas. El objetivo de este trabajo fue optimizar el protocolo de tratamiento de los eritrocitos humanos con estos dos copolímeros para mejorar su hemocompatibilidad. Para ello se estudió la influencia del tiempo de incubación de eritrocitos humanos con los dos copolímeros, analizándose las alteraciones producidas sobre la morfología y la capacidad de agregación eritrocitaria, utilizando técnicas de microscopía y análisis digital de imágenes. Se observó que los eritrocitos tratados con ambos copolímeros, si bien no sufrieron cambios en su morfología celular, presentaron alteraciones en la distribución del tamaño y en la estructura de sus agregados. En particular se observó un aumento del porcentaje de células individuales y una disminución del porcentaje de grandes agregados. Este efecto fue más significativo con el aumento del tiempo de incubación, siendo mayor para el Semo B86. Estos resultados permiten comprender mejor la interacción policatión/membrana y optimizar el protocolo de tratamiento de eritrocitos con estos compuestos, pudiéndose extender a otros copolímeros de estructuras similares con diferentes aplicaciones terapéuticas.

The effect of casein concentration, Ca2+ concentration, temperature and pH on the amount and size of protein aggregates (fines) in the whey produced by enzimic coagulation of nonfat milk was studied in laboratory conditions. Casein concentrations about 0.3 g/L showed a minimal amount of caseins in the whey, with presence of small aggregates of casein micellles. Ca2+ concentrations higher than 5 mM were neccesary to reduce the whey protein to a minimum constituted by protein particles smaller than casein micelles. The coagulation temperature, in the 35 - 45oC range, produced almost no variations in the whey proteins. The obtention of a minimum amount of whey proteins was possible only in a narrow pH range around 6.4. These results pointed to casein concentration and pH as important variables to be controlled in connection with the process yield.