Separación de poblaciones de glóbulos rojos senescentes por gradientes preformados de Percoll / Separation of populations of senescent red blood cells by Percoll performed gradients

Los mecanismos que determinan la senescencia de los eritrocitos han sido extensamente estudiados, sin embargo, no se han logrado conclusiones definitivas debido a la ausencia de una técnica que permita el aislamiento de grupos etáreos bien definidos. Los métodos más comúnmente empleados se basan en el aumento de densidad de los eritrocitos durante el envejecimiento. En este trabajo desarrollamos una técnica para la separación de glóbulos rojos de distintas edades empleando gradientes preformados de Percoll, un polímero sintético con propiedades fisicoquímicas adecuadas para trabajar con células vivas. En las suspensiones eritrocitarias obtenidas se realizaron determinaciones hematológicas, actividades de enzimas antioxidantes y el ensayo de eritrofagocitosis. Los valores de los parámetros hematológicos evaluados fueron significativamente mayores en las suspensiones de glóbulos rojos jóvenes. Las actividades enzimáticas mostraron una disminución de la capacidad antioxidante en las poblaciones de eritrocitos senescentes. Este proceso favorecería la interacción de los hematíes envejecidos con las células fagocíticas, demostrada median­te el ensayo de eritrofagocitosis. Los resultados obtenidos indican que el método de gradientes de Percoll permite una adecuada separación de las suspensiones eritrocitarias de distintas edades, con una eficiencia comparable a la observada en la técnica de centrifugación diferencial considerada de referencia.

The mechanisms that determine the senescence of the erythrocytes have been extensively studied; however. definitive conclusions have not been achieved mainly because of the lack of a technique that allows the isolation of well-defined etarian groups. The methods most commonly used for separating erythrocytes from different ages are based on the increase in density that these cells present during their aging. In the present work we have developed a technique for obtaining red blood cells from different ages using Percoll preformed gradients, a synthetic polymer with adequate physic-chemic properties to work with lives cells. In the erythrocytes suspensions we have made hematological determinations. activities of antioxidants enzymes and the essay of erythrophagocytosis. The values of the hematological parameters were significantly higher in the suspensions of young red blood cells. In the measurements of the enzymatic activity we observed a decrease of the antioxidant capacity in the populations of senescent erythrocytes. This process would promote the interaction between the old erythrocytes and the phagocyte cells, demonstrated by the erythrophagocytosis essay. The results obtained indicate that the method Percoll density gradients allows an appropriate separation of the erythrocytes suspensions of different ages with a comparable efficiency to that observed in the technique differential centrifugation, considered as reference.

Determinación de IgG en eritrocitos senescentes por un ensayo enzimático con consumo de antiglobulina / Determination of senescent red blood cells bound IgG using an enzyme test based an the consuption of antiglobulin

Durante el envejecimiento de los GR se acumula IgG autóloga sobre la membrana eritrocitaria. Esta IgG esta dirigida a un neo antígeno de senescencia, ubicado en la proteína banda 3. El objetivo de este trabajo fue determinar pequeñas cantidades de IgG en la membrana del GR utilizando un inmunoensayo con antiglobulina conjugada con una enzima. Las suspensiones de GRSe y GRJ fueron incubadas con anti-IgG humana conjugada con fosfatasa alcalina. Se transfirieron alícuotas a microplacas sensibilizadas con IgG humana. Se agregó el sustrato de la enzima y se midió la IgG libre. Las concentraciones de IgG unida a la membrana eritrocitaria en GRSe (13.31 x 10-4(g/(L(1.57 x 10-4) fueron significativamente mayores (p(0.0001) que los valores observados en GR (3.35 x 10-4(g/(L(1.39 x 10-4). Los resultados obtenidos indican que esta metodología constituye una herramienta útil para determinar pequeñas contidades de IgG unida a la membrana eritrocitaria.

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 difractometric 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 antigenic expression could lead to physiological phagocytosis.

Autoimmune hemolytic anemia.

Immune hemolytic anemia can be either isoimmune or autoimmune. Autoimmune hemolytic anemias (AIHA) consist of group of disorders whose common characteristics are the presence of an antibody which in turn causes short red blood cell (RBC) life. The rate and site of hemolysis and hence the clinical manifestations depends on the type of antibody attached and its propensity to fix complement. Antibodies of the IgG class are most commonly responsible for AIHA in children. Rh erythrocyte antigen is involved in more than 70% of cases. Since the antibody has its maximal activity at 37 degrees C, the resultant hemolysis is called warm antibody induced hemolytic anemia. This is a severe life threatening condition, the clinical features are: sudden onset of pallor, jaundice and dark urine. The cornerstone of diagnosis is a positive Coomb's antiglobulin test in the presence of hemolysis. Coomb's test has false negative and false positive rates in about 2-4% and 8% of all cases respectively. The modalities for treatment of warm AIHA include blood transfusion, steroid therapy, intravenous gammaglobulin, plasma-pheresis and splenectomy. The choice depends on the severity of the disease and child's response to therapy.