Alelos RHD nulos en pacientes RhD negativo de una población hospitalaria de la ciudad de Rosario / RHD null alleles in RhD negative patients from a hospital of Rosario

El fenotipo RhD negativo en la población caucásica es causado por una deleción completa del gen RHD. Sin embargo, han sido reportadas regiones específicas de este gen en individuos RhD negativo de diferentes grupos étnicos. El objetivo de este trabajo fue investigar la presencia de alelos RHD nulos en pacientes RhD negativo que concurrieron al Hospital Provincial del Centenario. Se tipificaron 12672 individuos y se seleccionaron las muestras RhD negativo halladas. Se determinó el fenotipo Rh completo y posteriormente se investigó la presencia del gen RHD utilizando una estrategia de PCR multíplex. En las muestras que presentaron fragmentos RHD específicos se realizaron reacciones de PCR alelo específicas para determinar el origen de los exones. Se encontraron 653 (5.15%) muestras RhD negativo. Cincuenta y cinco (8.42%) presentaban al menos el antígeno RhC o RhE. Los estudios moleculares permitieron detectar 7 alelos RHD Psi, 5 alelos híbridos RHD-CE(3-7)-D, 2 alelos híbridos RHD­CE(3-9)-D y 1 alelo nuevo RHD (46 T>C). La frecuencia de individuos RhD negativo en la población estudiada fue significativamente menor a la reportada en caucásícos. Los resultados moleculares obtenidos indican que 2.30% (15/653) de los individuos que no expresan el antígeno D son portadores de alelos RHD nulos. Los alelos RHD-CE(3-7)-D, RHD-CE(3-9)-D y RHD (46 T>C) están presentes únicamente en individuos RhD negativo que expresan los antígenos RhC y/o RhE con una frecuencia del 14.50% (8/55). Por otro lado, el alelo RHD Psi está asociado exclusivamente al fenotipo dccee, siendo el 1.17% (7/598) de estos individuos portadores del pseudogen RHD Psi. Estos hallazgos señalan la importancia del estudio del polimorfismo molecular del locus RH para el desarrollo de estrategias de tipificación de ADN confiables, que permitan realizar la genotipificación RHD prenatal y optimizar la selección de unidades a transfundir en los Bancos de Sangre.

The RhD negative phenotype in Caucasians is mainly caused by a complete deletion of the RHD gene. However, specific regions of the RHD gene in RhD negative individuals have been reported in different ethnic groups. The purpose of this study was to analyse the presence of silent RHD alleles in RhD negative patients concurring to the Hospital Provincial del Cen­tenario. Blood samples from 12672 individuals were studied and the RhD negative phenotypes were selected. Initially, the complete Rh phenotype was determined and DNA samples were screened using a multiplex PCR strategy to detect the presence of an RHD allele. Samples carrying RHD specific fragments were further studied by RHD exon scanning with allele specific PCR. 653 samples out of the 12672 (5.15%) were found RhD negative. Within this group, 8.42 % were either RhC positive or RhE positive. Molecular studies detected 7 RHD Psi alleles, 5 RHD-CE(3-7)-D hybrid alleles, 2 RHD-CE(3-9)-D hybrid alleles and 1 RHD (46 T>C) novel allele.The frequency of RhD negative individuals observed in the population studied was lower than that reported for Caucasians. Molecular analysis showed that 2.30% (15/653) of the individuals with no expression of the D antigen carry RHD null alleles. RHD­CE(3-7)-D, RHD-CE(3-9)-D and RHD (46 T>C) alleles are present only in individuals expressing either RhC or RhE with a frequency of 14.55% (8/55). The RHD Psy is associated with the dccee phenotype and 1.17% (7/ 598) of these individuals carries the RHD Psi pseudogen. These findings highlight the importance of studying the molecular polymorphism of the RH locus so as to develop reliable DNA typing strategies.

Effects of aging on antioxidant response and phagocytosis in senescent erythrocytes.

Red blood cell (RBC) aging is a complex process affected by immunological and biochemical parameters. In this work we studied the antioxidant response in RBC of different ages. We also investigated their interaction with peripheral blood monocytes. Anticoagulated blood samples from 19 O RhD+ volunteers' donors were processed. Young (Y) RBC and Senescent (Se) RBC were obtained by self-formed gradients of Percoll. The fractionation of the erythrocytes suspensions was demonstrated by statistically significant density-related changes in hematological determinations. Activities of glucose-6-phosphate dehydrogenase (G6PD), of soluble NADH-cytochrome b5 reductase (b5Rs) and membrane-bound b5R (b5Rm) were determined spectrophotometrically. The interaction between monocytes and different RBC suspensions was evaluated by the erythrophagocytosis assay. The G6PD and b5Rm activities in SeRBC were significantly lower than that observed in YRBC. No differences were found in the b5Rs of both groups. We observed an increased rate of erythrophagocytosis the SeRBC compared to YRBC. The decline in the activities of G6PD and b5Rm would indicate a decrease in the antioxidant response associated to RBC aging. These findings would signify that the oxidative changes of membrane occurring during the life span of the RBC might be relevant in the process of removal of SeRBC from the circulation.

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.