RESUMO
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
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
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.