Standardization of a multiplex assay to identify weak D types in a mixed-race Brazilian population.

RH allele variability is caused by several types of variants, resulting in altered RhD and RhCE phenotypes. Most of the weak D phenotypes in European-derived populations are weak D types 1, 2, or 3, which are not involved in alloimmunization episodes. However, the Brazilian population is racially diverse, and the accuracy of molecular and serologic tests developed in recent years has allowed for the identification of other RH variants, that are common in the Brazilian population, such as weak D type 38 or weak partial 11, the latter involved in alloimmunization cases. Furthermore, patients with these two weak D variants must be transfused with D- red blood cell units, as do patients with weak D type 4 or DAR, which are also common D variants in Brazil. Weak D type 38 and weak partial 11 can be serologically misclassified as weak D types 1, 2, or 3 in patients, based on European experience, or as D- in donors. Additionally, pregnant women may unnecessarily be identified as requiring Rh immune globulin. RhCE phenotypes are reliable indicators of RhD variants. For individuals with the Dce phenotype, the preferred approach is to specifically search for RHD*DAR. However, when encountering DCe or DcE phenotypes, we currently lack a developed method that assists us in rapidly identifying and determining the appropriate course of action for the patient or pregnant woman. Two multiplex assays were proposed: one for the identification of RHD*weak partial 11, RHD*weak D type 38, and RHD*weak D type 3 and another for RHD*weak D type 2 and RHD*weak D type 5. The multiplex assays were considered valid if the obtained results were equivalent to those obtained from sequencing. Expected results were obtained for all tested samples. The proposed multiplex allele-specific polymerase chain reaction assays can be used in the molecular investigation of women of childbearing age, patients, and blood donors presenting a weak D phenotype with DCe or DcE haplotypes in a mixed-race population, such as Brazil.

-318C/T polymorphism of the CTLA-4 gene is an independent risk factor for RBC alloimmunization among sickle cell disease patients.

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) molecule is expressed on T-lymphocyte membrane and negatively influences the antigen-presenting process. Reduced expression of CTLA-4 due to gene polymorphisms is associated with increased risk of autoimmune disorders, whose physiopathology is similar to that of post-transfusion red blood cell (RBC) alloimmunization. Our goal was to evaluate if polymorphisms of CTLA-4 gene that affect protein expression are associated with RBC alloimmunization. This was a case-control study in which 134 sickle cell disease (SCD) patients and 253 non-SCD patients were included. All patients were genotyped for the polymorphisms 49A/G and -318C/T of CTLA-4 gene. The genotype frequency of -318C/T differed significantly between alloimmunized and nonalloimmunized SCD patients, irrespective of clinical confounders (p = .016). SCD patients heterozygous for -318T allele presented higher risk of alloantibody development (OR: 5.4, CI: 1.15-25.6). In conclusion, the polymorphism -318C/T of CTLA-4 gene is associated with RBC alloimmunization among SCD patients. This highlights the role played by CTLA-4 on post-transfusion alloantibody development.

Screening for the SMIM1*64_80 del Allele in blood donors in a population from Southern Brazil.

BACKGROUND AND OBJECTIVES: Serological screening for the Vel- phenotype is complex given the large individual variation in the levels of expression of the Vel antigen, and the polyclonal anti-human sera of immunised persons, when available, show heterogeneous reactivity levels. Studies of the SMIM1 gene have enabled the development of several molecular methodologies that will be crucially important for the screening of different populations, including Brazilians. To evaluate the deletion of 17 bp in the SMIM1 gene in a population from the south of Brazil, 448 unrelated blood donors from 7 regions comprising the haemotherapy network in the state of Santa Catarina were evaluated between August 2011 and March 2014. MATERIALS AND METHODS: DNA samples from these donors were analysed employing a 5' nuclease real-time polymerase chain reaction (PCR) assay targeting the 17 bp deletion in the SMIM1 gene. RESULTS: Among the 448 samples analysed, 10 (2·23%) harboured the 17 bp deletion of the gene SMIM1, and all were heterozygote for the SMIM1*64_80 del allele. CONCLUSION: The allelic frequency found differed from those observed in other Caucasian populations. This difference can be explained by the ethnic make-up of each Caucasian population. The data obtained are important to characterise the correct phenotype of the donor as the serological assay results are not reliable due to variations in the expression intensity of the Vel antigen in heterozygote donors for the SMIM1*64_80 del allele. Moreover, the tool used in this study is of great value for identifying a donor Vel- phenotype and supplying a possible need for transfusion.

RHD allelic identification among D-Brazilian blood donors as a routine test using pools of DNA.

BACKGROUND: RHD alleles leading to a reduced expression of D antigen of the red blood cell (RBC) surface may be erroneously typed as D- by serology and may cause anti-D immunizations when transfused to recipients. METHODS: To determine the occurrence of such alleles among apparent D- blood donors, molecular typing was implemented as a routine test using a pool of DNA. A total of 2,450 pretyped D- samples were tested in pools of 10 for the RHD-specific polymorphism in intron 4 and exon 7. Samples in polymer chain reaction (PCR) positive pools were individually reevaluated by exon-specific PCRs, sequencing, and serologic methods. RESULTS: Among 2,450 serologically D- blood donor samples tested, 101 (4.1%) carried the RHD gene. Nonfunctional RHD (RHDψ, RHD*CE(2-9)-D, and RHD*CE(3-7)-D), different weak D alleles such as RHD*weak D type 1, RHD*weak D type 4.3, RHD*weak D type 5, RHD*weak D type 38, and RHD*DEL were identified. CONCLUSION: We employed a PCR-based assay for RHD as a routine test using pools of ten DNA blood donor samples. The integration of RHD genotyping into the routine screening program using pools of DNA samples was straightforward. As a consequence, 19 (0.8%) blood donors carrying a weak D and Del phenotypes with the potential of causing anti-D immunizations in recipients were reclassified as D+. For each population, it would be necessary to adapt the RHD genotyping strategy to the spectrum of prevalent alleles.