RESUMO
Determination of the erythrocyte lifespan is a complex process affected by many cellular parameters. In the present study we measured and characterised the red blood cell (RBC) membrane proteins, mainly band 3, and quantified membrane-bound IgG in senescent RBC (SeRBC) and young RBC (YRBC). We also investigated, through a functional assay, the interaction between SeRBC and peripheral blood monocytes. We applied this erythrophagocytosis assay to study the phagocytosis of desialysed RBC. The results obtained showed no changes in the protein content between SeRBC and YRBC and no differences when examining membrane proteins by SDS-PAGE. Then, considering that the accumulation of autologous IgG on RBC membrane provides a direct mechanism for the removal of SeRBC, we measured the IgG content of intact RBC using an enzyme-linked anti-immunoglobulin test finding that the number of IgG molecules bound to SeRBC was significantly higher than that observed for YRBC. The increase observed in the percentage of erythrophagocytosis with SeRBC and sensitised RBC (SRBC) confirmed the involvement of autologous IgG in the selective removal of erythrocytes. We also observed a higher percentage of monocytes with phagocytosed and adherent RBC (AM) obtained with neuraminidase-treated RBC than those obtained with YRBC. This finding suggests that a decrease in sialic acid content of SeRBC may be involved in physiological erythrophagocytosis.
Assuntos
Envelhecimento Eritrocítico/imunologia , Envelhecimento Eritrocítico/fisiologia , Imunoglobulina G/sangue , Monócitos/fisiologia , Membrana Eritrocítica/imunologia , Membrana Eritrocítica/metabolismo , Humanos , Técnicas In Vitro , Fagocitose , Ácidos Siálicos/sangueRESUMO
BACKGROUND AND OBJECTIVES: The Rh system is genetically controlled by the homologous RHD and RHCE genes that encode the RhD and RhCcEe polypeptides, respectively. Deletions, point mutations and rearrangements between both genes are responsible for the great polymorphism of this system. The aim of this work was to analyse the genetic basis of a Dc- phenotype. MATERIALS AND METHODS: DNA samples from the Dc- propositus and family members were obtained from peripheral blood. RHCE intron 4-exon 5 and RH exons 4, 5, 6 and 7 were analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Exon 9 was studied by PCR-sequence-specific primers (SSP). The RH locus was further analysed by using a PCR designed for a hybrid allele. RESULTS: No RHCE-specific fragments were found when analysing exons 5, 6 and 7 of the RH locus from the propositus' DNA, while exons 4 and 9 of both RH genes were present. CONCLUSIONS: The results obtained indicated that the Dc- phenotype is encoded by a novel RHCE-D(5-7/8)-CE hybrid allele.
Assuntos
Alelos , Sistema do Grupo Sanguíneo Rh-Hr/genética , Análise Mutacional de DNA , Epitopos , Éxons , Saúde da Família , Glicoproteínas/genética , Humanos , Polimorfismo Genético , Sistema do Grupo Sanguíneo Rh-Hr/imunologiaRESUMO
There is a decrease in the percentage contribution of a heavy density fraction of red blood cells to whole blood with increasing age. The aim of this study was to investigate in the young and elderly the interaction between monocytes and different erythrocyte suspensions: senescent red blood cells, erythrocytes stored with or without serum, and desialylated red blood cells. The results obtained with senescent red blood cells and erythrocytes stored with serum show the involvement of autologous IgG in the selective removal of erythrocytes. These values were higher in elderly individuals, indicating that this process increases with age. Our observation suggest that desialylation is not involved in the increased removal of erythrocytes observed in elderly individuals.
Assuntos
Senescência Celular , Eritrócitos/fisiologia , Monócitos/metabolismo , Fagocitose/fisiologia , Adulto , Fatores Etários , Idoso , Preservação de Sangue , Eritrócitos/citologia , Humanos , Pessoa de Meia-IdadeRESUMO
The aim of this work was to investigate the presence of the RHD gene in fetal cells obtained from amniotic fluid. We studied 65 samples of amniotic fluid, 11 from RhD-negative mothers sensitized with anti-D alloantibodies. The fetal origin of the DNA was confirmed with the analysis of 1 VNTR locus and 3 STR loci in DNA samples from amniotic fluid and maternal blood. The RHD genotyping was performed in non-contaminated samples (n=62) using a multiplex polymerase chain reaction strategy that yields three amplification products from RhD-positive phenotypes (intron 4 of both RHCE and RHD genes and exon 10 of the RHD gene) and I DNA fragment from RhD-negative phenotypes (intron 4 of the RHCE gene). We genotyped 54 RhD-positive fetuses (8 from RhD-negative sensitized mothers) and 8 RhD-negative fetuses (3 from RhD-negative sensitized mothers). The fetal DNA genotyping allows the diagnosis, from a single amniocentesis, of fetuses at real risk of hemolytic disease of the newborn. When the fetus is determined to be RhD-negative invasive procedures can be avoided.
Assuntos
Eritroblastose Fetal/diagnóstico , Sistema do Grupo Sanguíneo Rh-Hr/análise , Líquido Amniótico/química , DNA , Eritroblastose Fetal/genética , Feminino , Feto , Idade Gestacional , Humanos , Recém-Nascido , Repetições Minissatélites , Reação em Cadeia da Polimerase , Gravidez , Diagnóstico Pré-Natal , Sistema do Grupo Sanguíneo Rh-Hr/genética , Fatores de Risco , Sensibilidade e EspecificidadeRESUMO
Human red blood cells (RBC) have a well-defined lifespan of 120 days affected by many cellular parameters. The aim of the present study was to investigate through a functional assay the effect of some factors in the interaction of erythrocytes with monocytes: heat rigidification, equilibration at different pH and desialyzation. We also studied the interaction between stored RBC and peripheral blood monocytes with this functional erythrophagocytosis assay. Blood samples from 30 volunteer donors were investigated. 1) Senescent (Se) and Young (Y) RBC were obtained by differential centrifugation. 2) Erythrocyte suspensions: Aliquots of each sample were subjected to the following treatments: a) Rigidification by heat (RRBC), b) Equilibration at different pH (5.34, 6.30, 7.33, 9.20) and c) Desialyzation with neuraminidase and trypsin. The functional assay was performed incubating monocytes obtained by glass adherence with these suspensions of RBC. Whole blood samples (n = 20) were stored during different periods of time (0, 7, 14, 21, 28, 35 and 42 days). The erythrophagocytosis assay was performed during six weeks incubating isologous monocytes with RBC from every unit. Negative and positive controls were performed using non sensitized (NSRBC) and sensitized with IgG anti-RhD (SRBC) red cells. The percentage of active monocytes (AM) obtained were: 1) YRBC: 2.8 +/- 0.9 and SeRBC: 17.5 +/- 2.1; 2a) RRBC: 3.0 +/- 0.9; 2b) 10.9 +/- 0.9, 15.5 +/- 0.8, 3.1 +/- 1.0, 4.0 +/- 1.1; 2c) 11.1 +/- 1.4 and 3.9 +/- 1.0; SRBC 32.1 +/- 1.7 and NSRBC: 2.8 +/- 1.5. The % of AM with SeRBC was higher (p < 0.001) than those obtained with NSRBC. The data of AM with RRBC were significantly lower (p < 0.001) than those obtained with SeRBC and SBRC, indicatingthat heat rigidification of RBC does not increase phagocytosis by monocytes. The values of AM obtained from the suspensions of erythrocytes equilibrated at different pH indicate that the acidification of RBC increases the interaction with monocytes. The % AM with neuraminidase treated RBC was higher than those observed with YRBC and NSRBC (p < 0.001). No modifications were observed with trypsin treated RBC. These results suggest that the loss of sialic acid may be involved in the physiological phagocytosis. The values of AM of stored whole blood were: 2.3 +/- 1.3, 2.7 +/- 1.3, 4.4 +/- 1.6, 6.7 +/- 1.2, 9.6 +/- 1.0, 11.7 +/- 0.8 and 13.0 +/- 1.2. The results showed a significant increase in the % of AM as a function of the preservation time from 2,3 +/- 1,3 for the first day to 13,0 +/- 1,2 for the 42nd day (p < 0.001). The data obtained in this ex vivo model show a significant increase (p<0.001) in the phagocytosis of RBC equilibrated at low pH, desialinized (greater than 80%) with neuraminidase and stored for over 28 days. These factors would be involved in erythrocyte removal via phagocytosis during tissular homeostasis.
Assuntos
Senescência Celular , Eritrócitos/fisiologia , Eritrócitos/citologia , Humanos , Concentração de Íons de HidrogênioRESUMO
The aim of this paper is to evaluate the erythrophagocytosis assay (EA) in patients with autoimmune hemolytic anemia (AIHA). Direct antiglobulin test (DAT), indirect antiglobulin test (IAT) and EA were performed in blood samples from 46 patients with presumed AIHA. The EA was carried out incubating patients' erythrocytes and peripheral blood monocytes. A total of 200 monocytes were analysed to determine the percentage of active phagocytic cells (% APC). In 9 of these patients the applied treatment was evaluated by DAT, IAT and EA. In 14 transfusion requirements, the compatibility tests and EA were performed. For EA, patients' monocytes were incubated with erythrocytes from previously selected units sensitized with patients' sera. The % of APC was 32.1 +/- 1.7 in 35 patients with positive DAT and 17.8 +/- 1.3 in 11 patients with negative DAT. This last value was significantly higher than that with negative controls (3.7 +/- 0.3)(p < or = 0.01). As regards the applied treatment, patients with a successful response (n = 6) showed a significant decrease in the initial % APC (31.8 +/- 1.6 to 15.3 +/- 2.4; p < or = 0.05) while DAT and IAT remained positive. In those patients who required blood transfusion the compatibility tests were positive with all the units to be transfused, whereas the % APC varied for each one. Blood units were selected according to the lower % APC.
Assuntos
Anemia Hemolítica Autoimune/fisiopatologia , Eritrócitos/fisiologia , Fagocitose/fisiologia , Anemia Hemolítica Autoimune/diagnóstico , Anemia Hemolítica Autoimune/terapia , Transfusão de Sangue , Contagem de Células , Humanos , Fagócitos/fisiologiaRESUMO
The aim of this work was to determine the presence of the RHD gene in fetal cells obtained from amniotic fluid (AF). We studied 65 samples of AF, 11 from RhD- mothers sensitized with anti-D. The fetal origin of the DNA was confirmed with the analysis of 1 VNTR locus and 3 STR loci in DNA samples from AF and maternal blood. The RHD genotyping was performed in non contaminated samples (n = 62) using a multiplex PCR strategy that yields 3 amplification products from RhD+ phenotypes and 1 DNA fragment from RhD- phenotypes. We genotyped 54 RhD+ fetuses (8 from RhD- sensitized mothers) and 8 RhD- fetuses (3 from RhD- sensitized mothers). Fetal DNA genotyping allows the diagnosis, from a single amniocentesis, of fetuses at real risk of hemolytic disease of the newborn. When the fetus is determined to be RhD- all invasive procedures can be avoided.
Assuntos
Líquido Amniótico/imunologia , Eritroblastose Fetal/diagnóstico , Sistema do Grupo Sanguíneo Rh-Hr/genética , DNA/genética , Eletroforese em Gel de Ágar , Eritroblastose Fetal/genética , Feminino , Marcadores Genéticos , Genótipo , Humanos , Reação em Cadeia da Polimerase , GravidezRESUMO
The aim of this paper is to evaluate the erythrophagocytosis assay (EA) in patients with autoimmune hemolytic anemia (AIHA). Direct antiglobulin test (DAT), indirect antiglobulin test (IAT) and EA were performed in blood samples from 46 patients with presumed AIHA. The EA was carried out incubating patients erythrocytes and peripheral blood monocytes. A total of 200 monocytes were analysed to determine the percentage of active phagocytic cells (
APC). In 9 of these patients the applied treatment was evaluated by DAT, IAT and EA. In 14 transfusion requirements, the compatibility tests and EA were performed. For EA, patients monocytes were incubated with erythrocytes from previously selected units sensitized with patients sera. The
of APC was 32.1 +/- 1.7 in 35 patients with positive DAT and 17.8 +/- 1.3 in 11 patients with negative DAT. This last value was significantly higher than that with negative controls (3.7 +/- 0.3)(p < or = 0.01). As regards the applied treatment, patients with a successful response (n = 6) showed a significant decrease in the initial
APC (31.8 +/- 1.6 to 15.3 +/- 2.4; p < or = 0.05) while DAT and IAT remained positive. In those patients who required blood transfusion the compatibility tests were positive with all the units to be transfused, whereas the
APC varied for each one. Blood units were selected according to the lower
APC.
RESUMO
The aim of this work was to determine the presence of the RHD gene in fetal cells obtained from amniotic fluid (AF). We studied 65 samples of AF, 11 from RhD- mothers sensitized with anti-D. The fetal origin of the DNA was confirmed with the analysis of 1 VNTR locus and 3 STR loci in DNA samples from AF and maternal blood. The RHD genotyping was performed in non contaminated samples (n = 62) using a multiplex PCR strategy that yields 3 amplification products from RhD+ phenotypes and 1 DNA fragment from RhD- phenotypes. We genotyped 54 RhD+ fetuses (8 from RhD- sensitized mothers) and 8 RhD- fetuses (3 from RhD- sensitized mothers). Fetal DNA genotyping allows the diagnosis, from a single amniocentesis, of fetuses at real risk of hemolytic disease of the newborn. When the fetus is determined to be RhD- all invasive procedures can be avoided.
RESUMO
Rhesus D (RhD) typing is performed by agglutination methods; however, in clinical situations where these techniques cannot be performed, RhD DNA typing is an alternative approach. The Rh antigens are encoded by the RHD and RHCE genes. In RhD-negative individuals the RHD gene is absent or grossly deleted, but variations in the arrangement of the RH locus in different populations are emerging. The aim of this study was to analyse the gross organization of the RH genes in our population using a previously described multiplex polymerase chain reaction (PCR) method with some modifications. We studied 253 DNA samples from Argentinian blood donors, 15 samples with a reduced expression of the D antigen and 1 Dc- phenotype. We evaluated the clinical utility of this method to ascertain the RhD antigen in 10 patients with warm-type autoimmune haemolytic anaemia (AIHA) and 14 samples of amniotic fluids. All Rh phenotypes were properly characterized and no discrepancies with serological typing were found. Analyses performed in the Dc- phenotype suggest the presence of a hybrid RHCE-RHD gene. DNA typing confirmed the RhD-negative type of one AIHA sample in which serological tests were inconclusive. Foetal DNA typing correctly indicated the RhD in every foetus. VNTR (variable number of tandem repeats) and STR (short tandem repeats) analysis detected maternal contamination in two amniocentesis samples and confirmed the foetal origin of 12. This multiplex PCR strategy is suitable for RhD determination in clinical situations in which serological typing cannot be accomplished with its usual ease.
Assuntos
Anemia Hemolítica Autoimune/sangue , DNA/análise , Sistema do Grupo Sanguíneo Rh-Hr/genética , Alelos , Líquido Amniótico/química , Líquido Amniótico/fisiologia , Teste de Coombs , Primers do DNA/química , Glicoproteínas/genética , Humanos , Repetições Minissatélites/genética , Fenótipo , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Testes Sorológicos , Sequências de Repetição em Tandem/genéticaRESUMO
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.
Assuntos
Comunicação Celular/fisiologia , Envelhecimento Eritrocítico/fisiologia , Monócitos/fisiologia , Antígenos/biossíntese , Viscosidade Sanguínea , Envelhecimento Eritrocítico/imunologia , Humanos , Fagocitose/fisiologia , ReologiaRESUMO
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.
RESUMO
Existe amplia evidencia de que el cáncer está asociado con anormalidades en la regulación génetica expresada en la superficie de la membrana celular. El 80 por ciento de los individuos son capaces de secretar los antígenos ABH en saliva y otras secreciones. La presencia de estas sustancias está controlada por un gen que puede adoptar dos formas alélicas: SE dominante y SE recesiva. El objetivo de este trabajo fue investigar la relación entre la expresión antigénica ABH en célkulas de descamación urotelial y el carácter secretor en pacientes con cáncer de vejiga. Se examinaron 33 pacientes con tumores de vejiga clasificados en superficiales y profundos y una población de 40 individuos normales. Se investigó el carácter secretor en saliva y la expresión de los antígenos ABH uroteliales en sedimentos urinario. Se empleó para estos estudios la técnica de inhibición de la aglutinación. En la población normal todos expresaron los antígenos ABH en células de sedimento urinario y sólo el 80 por ciento presentó dichos antígenos en sus secreciones. En los pacientes con cáncer de vejiga el 30,31 por ciento resultó no secretor y de ellos el 70 por ciento presentó deleción antigénica ABH en sedimento urinario con mayor incidencia de tumores profundos. Nuestros resultados indicarían que los pacientes con cáncer de vejiga no secretores desarrollarían tumores con mayor grado de infiltración respecto de los pacientes secretores(AU)
Assuntos
Humanos , Neoplasias da Bexiga Urinária/urina , Neoplasias da Bexiga Urinária/sangue , Isoantígenos/isolamento & purificação , Saliva/metabolismo , Antígenos de Grupos Sanguíneos/análiseRESUMO
Existe amplia evidencia de que el cáncer está asociado con anormalidades en la regulación génetica expresada en la superficie de la membrana celular. El 80 por ciento de los individuos son capaces de secretar los antígenos ABH en saliva y otras secreciones. La presencia de estas sustancias está controlada por un gen que puede adoptar dos formas alélicas: SE dominante y SE recesiva. El objetivo de este trabajo fue investigar la relación entre la expresión antigénica ABH en célkulas de descamación urotelial y el carácter secretor en pacientes con cáncer de vejiga. Se examinaron 33 pacientes con tumores de vejiga clasificados en superficiales y profundos y una población de 40 individuos normales. Se investigó el carácter secretor en saliva y la expresión de los antígenos ABH uroteliales en sedimentos urinario. Se empleó para estos estudios la técnica de inhibición de la aglutinación. En la población normal todos expresaron los antígenos ABH en células de sedimento urinario y sólo el 80 por ciento presentó dichos antígenos en sus secreciones. En los pacientes con cáncer de vejiga el 30,31 por ciento resultó no secretor y de ellos el 70 por ciento presentó deleción antigénica ABH en sedimento urinario con mayor incidencia de tumores profundos. Nuestros resultados indicarían que los pacientes con cáncer de vejiga no secretores desarrollarían tumores con mayor grado de infiltración respecto de los pacientes secretores