Molecular analysis of the RHD pseudogene by duplex real-time polymerase chain reaction.

OBJECTIVES: In this study, we aimed to present a strategy for the detection of the RHD pseudogene (RHDψ) based on a real-time polymerase chain reaction (PCR) assay. BACKGROUND: The D-negative phenotype is associated with many genetic alterations. In populations with African ancestry, this phenotype commonly results from the silent variant RHDψ. The evaluation of RHDψ is essential for correct inference of the RhD phenotype in order to avoid false-positive results. METHODS: We utilised a new method for the simultaneous detection of RHDψ and a fragment from exon 5 of the wild-type RHD gene based on duplex real-time PCR assay. RESULTS: The PCR assay allowed specific detection of RHDψ. There was complete agreement between the results generated by the new test and the results generated by molecular analysis performed using end-point PCR methods previously described. CONCLUSIONS: The assay developed is easy to execute and presents the potential for routine use at blood banks and other associated facilities where it is desired to determine the presence of RHDψ.

Effect of red blood cell preservation by droplet freezing with non-permeable cryoprotective agents in blood group antigen reactivity.

BACKGROUND: In the last few decades, various red blood cell (RBC) freezing techniques have been developed and improved to enable the preservation of erythrocytes for future use in pre-transfusion tests in reference immunohaematology laboratories. However, not all these techniques have been sufficiently evaluated for the preservation of blood group antigens. OBJECTIVES: In this study, we evaluated the antigenic pattern of RBCs preserved by droplet freezing in liquid nitrogen in a blood bank context. METHODS: Blood samples were evaluated for the reactivity of blood group antigens after droplet freezing using the non-permeable cryoprotective agent polyvinylpyrrolidone (PVP) and sucrose-dextrose (S + D) solutions. RESULTS: No qualitative changes were observed in RBC reactivity after freezing and thawing for the antigens Fyb , Leb , C, E, Cw , Lua , Lub , Kpa , Kpb and Dia . However, cryopreservation using PVP resulted in a significant increase in reactivity of Fyb antigen on comparing fresh and frozen samples (P < 0·001). CONCLUSION: The establishment of detailed protocols for cryopreservation of RBCs, which take into account the maintenance of antigenic characteristics, is necessary to increase security in pre-transfusion testing using frozen RBCs.

Noninvasive fetal RHD genotyping from maternal plasma in an admixed Brazilian population.

We evaluated the efficacy of noninvasive fetal Rhesus D (RHD) genotyping from maternal plasma in a highly admixed population. Fifty-five blood samples from RhD-negative pregnant women from Brazil were processed for extraction of cell-free plasma DNA. Real-time PCR was performed to amplify segments of exons 5 and 7 from the RHD gene, as well as for detection of the SRY gene to confirm the presence of fetal DNA. Fetal genotyping results were compared with the RhD phenotype determined from newborn cord blood samples obtained at birth. Thirty-two samples were RHD-positive, 18 were RHD-negative and 5 were inconclusive due to amplification of only one RHD exon. In 43 samples, the fetal RHD genotype was compared to the neonatal RhD phenotype, and only one result was discordant, due to false-negative serology. There was one false SRY genotyping negative result. We conclude that noninvasive fetal RHD genotyping from maternal blood provides accurate results and suggests its viability as a clinical tool for the management of RhD-negative pregnant women in an admixed population.

Stability of family interaction from ages 6 to 18.

Research has demonstrated the stability of juvenile offending during childhood and adolescence but generally has not focused on the continuity of family interactions associated with juvenile offending. The present report focused on the stability of several family interaction events and attributes (i.e., physical punishment, communication, supervision, positive parenting, and parent-child relationship) for a large sample of male adolescents and their primary caretakers, drawn from a multiyear longitudinal study that represented middle childhood through late adolescence (ages 6-18). We also assessed the impact of ethnicity, family composition, teenage motherhood, and youth delinquency on these interactions. Test-retest correlations and growth-curve analyses were used to assess relative and absolute stability of the interactions, respectively. As predicted, relative stability of family interaction was high. There was an absolute change in scores of physical punishment (decreased) compared to poor supervision and low positive parenting (both increased), whereas poor communication and bad relationship with the caretaker did not measurably change with age. Single-parent families and families with teenage mothers experienced significantly worse interactions over time than did families consisting of two biological parents present in the household. These findings are discussed in relation to the development of juvenile offending.