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.

Dried blood spots of pooled samples for RHD gene screening in blood donors of mixed ancestry.

OBJECTIVES: In this study, we present a strategy for RHD gene screening based on real-time polymerase chain reaction (PCR) using dried blood spots of pooled samples. BACKGROUND: Molecular analysis of blood donors may be used to detect RHD variants among the presumed D-negative individuals. RHD genotyping using pooled samples is a strategy to test a large number of samples at a more reasonable cost. MATERIALS AND METHODS: RHD gene detection based on real-time PCR using dried blood spots of pooled samples was standardised and used to evaluate 1550 Brazilian blood donors phenotyped as RhD-negative. Positive results were re-evaluated by retesting single samples using real-time PCR and conventional multiplex PCR to amplify five RHD-specific exons. PCR-sequence-specific primers was used to amplify RHDψ allele. RESULTS: We devised a strategy for RHD gene screening using dried blood spots of five pooled samples. Among 1550 serologically D-negative blood donors, 58 (3.74%) had the RHD gene. The non-functional RHDψ allele was detected in 47 samples (3.02%). CONCLUSION: The present method is a promising strategy to detect the RHD gene among presumed RhD-negative blood donors, particularly for populations with African ancestry.

Human leukocyte antigen distribution and genomic ancestry in Brazilian patients with sickle cell disease.

Hematopoietic stem-cell transplantation (HSCT) is currently the only established curative treatment for sickle cell disease (SCD), but is limited by donor availability. Ethnicity is thought to have an impact on the complications experienced by patients that undergo HSCT and on the likelihood of identifying an human leukocyte antigen (HLA) matched donor. In the present study, we investigated the genomic ancestry and the distribution of HLA allele groups in Brazilian patients with SCD, compared these HLA profiles to worldwide populations and evaluate the availability of HLA-matched donors. A broad intercontinental admixture of patients with SCD was observed, with African ancestry ranging from 6.7% to 93.4%. In a dendrogram based on HLA frequencies, Brazilian patients with SCD were included in a branch containing only populations with a significant African component. Among the 126 patients evaluated, 10 (8%) found a HLA-matched unrelated donor in a database of 18 134 donors. Self-reported white, brown and black matched donors were identified, and no significant difference in the percentage of compatible donors was observed between these ethnic groups. Our results show that Brazilian patients with SCD are very admixed, indicating that this group is a promising target for admixture mapping of genes involved in complications after HSCT. Additional studies may help to clarify the impact of the genetic diversity and admixture of these patients on the donor availability.