Distribution of the FYBES and RHCE*ce(733C>G) alleles in an Argentinean population: implications for transfusion medicine.

BACKGROUND: The understanding of the molecular bases of blood groups makes possible the identification of red cell antigens and antibodies using molecular approaches, especially when haemagglutination is of limited value. The practical application of DNA typing requires the analysis of the polymorphism and allele distribution of the blood group genes under study since genetic variability was observed among different ethnic groups. Urban populations of Argentina are assumed to have a white Caucasian European genetic component. However, historical and biological data account for the influence of other ethnic groups. In this work we analyse FY and RH blood group alleles attributed to Africans and that could have clinical implications in the immune destruction of erythrocytes. METHODS: We studied 103 white trios (father, mother and child, 309 samples) from the city of Rosario by allele specific PCRs and serological methods. The data obtained were analysed with the appropriate statistical test considering only fathers and mothers (n = 206). RESULTS: We found the presence of the FY*BES and RHCE*ce(733C>G) alleles and an elevated frequency (0.0583) for the Dce haplotype. The number of individuals with a concomitant occurrence of both alleles was significantly higher than that expected by chance. We found that 4.68% of the present gene pool is composed by alleles primarily associated with African ancestry and about 10% of the individuals carried at least one RH or FY allele that is predominantly observed among African populations. Thirteen percent of Fy(b-) subjects were FY*A/FY*BES. CONCLUSION: Taken together, the results suggest that admixture events between African slaves and European immigrants at the beginning of the 20th century made the physical characteristics of black Africans to be invisible nowadays. Considering that it was a recent historical event, the FY*BES and RHCE*ce(733C>G) alleles did not have time to become widespread but remain concentrated within families. These findings have considerable impact for typing and transfusion strategy in our population, increasing the pool of compatible units for Fy(b-) individuals requiring chronic transfusion. Possible difficulties in transfusion therapy and in genotyping could be anticipated and appropriately improved strategies devised, allowing a better management of the alloimmunization in the blood bank.

Use of the erythrophagocytosis assay for predicting the clinical consequences of immune blood cell destruction.

The erythrophagocytosis assay is a useful parameter to evaluate the immune erythrocyte destruction occurring in vivo. The aim of this work was to use this assay in: a) the diagnostic and therapeutic assessment of autoimmune haemolytic anaemia (AIHA), b) the selection of blood for immunized patients requiring transfusion and c) the prediction of the severity of haemolytic disease of the newborn (HDN). This assay was also used to study the physiological removal of senescent erythrocytes from the circulation. The erythrophagocytosis assay was carried out incubating different erythrocyte suspensions and peripheral blood monocytes. A total of 200 monocytes were analysed to determine the percentage of active monocytes (% AM). We have demonstrated the usefulness of the erythrophagocytosis assay in the diagnosis of AIHA, mainly in patients with a negative direct antiglobulin test. This assay is also more adequate than classic immunohaematologic tests to obtain a better evaluation of the patients' response to treatment. The erythrophagocytosis assay was performed in immunized patients requiring transfusion and allowed us to select the least incompatible units. This method showed a better correlation with the newborns' clinical outcome than serological tests in cases of HDN. This functional assay also indicated an increased rate of erythrophagocytosis of senescent erythrocytes compared with young erythrocytes.

The Dc(G48)e(s) haplotype is frequent among the Dce haplotypes within a white population.

BACKGROUND: The absence of hybrid Rhesus boxes denotes an RHD homozygous status and helps to detect the presence of Dce haplotypes instead of dce. RHCE exon 1 C48, characteristic of RHC alleles, and RHCE exon 5 G733, responsible for VS antigenicity, have been noted in many RHce alleles but it was not clearly established whether they occurred in the same allele and/or cosegregate together with RHD. STUDY DESIGN AND METHODS: Samples from 148 white trios (father, mother, and child) were studied. Rh phenotype was performed by hemagglutination. Hybrid Rhesus box, RHCE exon 1 G48C, RHCE exon 5 C733G, and RHC intron 2 polymorphisms were analyzed by polymerase chain reaction. Haplotypes were determined considering serologic, molecular, and segregation data. RESULTS: RHCE exon 1 C48 and RHCE exon 5 G733 were present in RHce alleles that cosegregated with RHD forming Dce haplotypes. Both transversions were not frequently found in the same RHce allele. Of the 33 Dce haplotypes, 16 (48.5%) had a C at position 48 [Dc(C48)e], 11 (33.3%) had a G at position 48 with a G at position 733 [Dc(G48)e(s)], 5 (15.2%) had a G at position 48 [Dc(G48)e], and 1 (3.0%) had a C at position 48 with a G at position 733 [Dc(C48)e(s)]. CONCLUSIONS: The results show four molecular backgrounds for the Dce haplotype and reflect the contribution of African alleles to the genetic pool of the population under study. The molecular characterization of Dce and its frequency distribution may develop a better understanding of the phylogeny of Rh haplotypes.

Investigación de anticuerpos del sistema ABO en sangre de cordón de recién nacidos: cuantificación de anti-A / Detection of ABO system antibodies in umbilical cord blood from newborn infants: quantitation of anti-A

El objetivo de este trabajo fue determinar la presencia de anticuerpos del sistema ABO em 119 muestras de sangre de cordón de recién nacidos de grupo 0, cuyas madres presentaban el mismo grupo. Posteriormente se titularon los anticuerpos anti-A mediante un método manual de cuantificación de la aglutinación, relacionándolos con los anticuerpos presentes en la madre respectiva. Los resultados obtenidos demuestran la presencia de anticuerpos del sistema ABO en sangre de cordón umbilical en un 93,3% de las muestras (n = 111). Del total de estas muestras con reacción positiva, el 96,4% (n = 107) permaneció activo después del tratamiento con 2-mercaptoetanol, evidenciando su naturaleza de iunmunoglobulina G. La cuantificación de los anticuerpos anti-A demostró una menor concentración en los bebés que en la madre respectiva. Además, se observó que los bebés con títulos altos correspondían a las madres que presentaban títulos mayores. El análisis estadístico (r de Spearman) indicó que existe una asociación entre ambas variables

Enfermedad hemolítica fetoneonatal: estudio prenatal del genotipo RHD / Feto-neonatal hemolytic disease: prenatal study of RHD genotype

El objetivo de este trabajo fue determinar la presencia del gen RHD en células fetales obtenidas de líquido amniótico por PCR. Se estudiaron 65 muestras de líquido amniótico, 11 de madres RhD negativas sensibilizadas con anti-D. Se confirmó el origen fetal del ADN analizando un locus VNTR y 3 loci STR en las muestras de ADN de líquido amniótico y sangre materna. En las muestras no contaminadas (n = 62) se realizó la genotipificación RHD utilizando una estrategia de PCR multiplex que permite la obtención de tres productos de amplificación en los fenotipos RhD positivos y sólo un fragmento de ADN en los fenotipos RhD negativos. Se genotipificaron 54 fetos RhD positivos (8 de madres RhD negativas sensibilizadas) y 8 fetos RhD negativos (3 de madres RhD negativas sensibilizadas). La genotipificación del ADN fetal permite diagnosticar con una única amniocentesis fetos en riesgo real de enfermedad hemolítica fetoneonatal y evitar la utilización de métodos invasivos en casos de fetos RhD negativos.