Frequency of Wra antigen and anti-Wra in Brazilian blood donors

AbstractBackgroundWra is a low-incidence antigen, which is antithetical to the high prevalence red blood cell antigen, Wrb. Anti-Wra is a naturally occurring antibody that is found in approximately 1–2% of blood donors. The aim of this study was to determine the frequency of Wra and anti-Wra in Brazilian blood donors.MethodsA total of 1662 Brazilian blood donors were molecularly analyzed using the SNaPshot methodology to determine the WR*A/B alleles and to predict the frequency of the Wra antigen. To detect the anti-Wra, samples from 1049 blood donors were analyzed using a gel test with Wr(a+) red blood cells. The serum was treated with dithiothreitol (DTT) to determine the immunoglobulin classes. Immunoglobulin (Ig)-G isotype classification was performed in a gel test using the IgG1/IgG3 card. A monocyte monolayer assay was employed to predict the clinical significance of IgG anti-Wra.ResultsOf the 1662 donors, only one sample had the DI*02.03 allele in heterozygous predicting the Wr(a+b+) phenotype. Anti-Wra was detected in 34 (3.24%) samples, 64.7% in females and 35.3% in males. Regarding the immunoglobulin class, eight (23.5%) cases of anti-Wra were classified as IgG and 26 (76.5%) as IgM. Of the eight cases of IgG anti-Wra, four were IgG1, two were IgG3 and three anti-Wra were not IgG3 or IgG1, and thus probably IgG2 or IgG4. The results of the monocyte monolayer assay showed that IgG anti-Wra might be of clinical significance.ConclusionThis study shows a very low frequency (0.06%) of the Wra antigen in Brazilian blood donors. Additionally, it shows that the frequency of anti-Wra in this population is higher than previously reported

A New Strategy To Identify Rare Blood Donors: Single Polymerase Chain Reaction Multiplex Snapshot Reaction For Detection Of 16 Blood Group Alleles.

As an alternative to phenotyping, large-scale DNA-based assays, which are feasible for high-throughput donor red blood cell typing, were developed for determination of blood group polymorphisms. However, high-throughput genotyping platforms based on these technologies are still expensive and the inclusion of single nucleotide polymorphisms and analysis of the alleles depend on the manufacturer's determination. To overcome this limitation and in order to develop an assay to enable the screening of rare donors, we developed a SNaPshot assay for analysis of nine single nucleotide polymorphisms related to antigens that are difficult to assess using conventional serology. The single polymerase chain reaction multiplex SNaPshot reaction was optimized to identify nine single nucleotide polymorphisms determining 16 alleles: KEL*3/KEL*4, KEL*6/KEL*7, DI*1/DI*2, DI*3/DI*4, YT*1/YT*2, CO*1/CO*2, DO*1/DO*2, DO*4, DO*5. We designed a single multiplex PCR with primers encompassing the blood group single nucleotide polymorphisms and performed an internal reaction with probe primers able to discriminate the alleles after fragment analysis. The SNaPshot assay was validated with 140 known alleles previously determined by PCR restriction fragment length polymorphism. We were able to simultaneous detect nine single nucleotide polymorphisms defining 16 blood group alleles on an assay based on a multiplex PCR combined with a single base extension using genomic DNA. This study demonstrates a robust genotyping strategy for conducting rare donor screening which can be applied in blood centers and could be an important tool for identifying antigen-negative donors and, therefore, for providing rare blood.12 Suppl 1s256-6

An easy and efficient strategy for KEL genotyping in a multiethnic population

BACKGROUND: The Kell blood group system expresses high and low frequency antigens with the most important in relation to transfusion including the antithetic KEL1 and KEL2; KEL3 and KEL4; KEL6 and KEL7 antigens. Kell is a clinically relevant system, as it is highly immunogenic and anti-KEL antibodies are associated with hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Although required in some situations, Kell antigen phenotyping is restricted due to technical limitations. In these cases, molecular approaches maybe a solution. This study proposes three polymerase chain reaction genotyping protocols to analyze the single nucleotide polymorphisms responsible for six Kell antithetic antigens expressed in a Brazilian population. METHODS: DNA was extracted from 800 blood donor samples and three polymerase chain reaction-restriction fragment length polymorphism protocols were used to genotype the KEL*1/KEL*2, KEL*3/KEL*4 and KEL*6/KEL*7 alleles. KEL*3/KEL*4 and KEL*6/KEL*7 genotyping was standardized using the NlaIII and MnlI restriction enzymes and validated using sequencing. KEL*1/KEL*2 genotyping was performed using a previously reported assay. RESULTS: KEL genotyping was successfully implemented in the service; the following distribution of KEL alleles was obtained for a population from southeastern Brazil: KEL*1 (2.2%), KEL*2 (97.8%), KEL*3 (0.69%), KEL*4 (99.31%), KEL*6 (2.69%) and KEL*7 (97.31%). Additionally, two individuals with rare genotypes, KEL*1/KEL*1 and KEL*3/KEL*3, were identified. CONCLUSION: KEL allele genotyping using these methods proved to be reliable and applicable to predict Kell antigen expressions in a Brazilian cohort. This easy and efficient strategy can be employed to provide safer transfusions and to help in rare donor screening.9910

Strategy to detect RhD variants in blood donors

Orientador: Lilian Maria de CastilhoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: O antígeno D é o mais imunogênico no sistema Rh. O antígeno D é muito polimórifco e certas mutações e/ ou deleções levam a um fenótipo fraco definido por uma baixa densidade antigênica e que requerem a realização de testes até a fase de anti-globulina humana (AGH) para sua detecção. Neste estudo foram avaliadas diferentes tecnologias e clones de anti-D para propor a estratégia sorológica mais adequada para detectar o maior número de variantes D em doadores de sangue. Métodos: Foram selecionadas 101 amostras de doadores de sangue brasileiros com diferentes expressões de D na rotina de doadores. Os testes foram realizados em leitura imediata (LI) com onze reagentes anti-D comercialmente disponíveis em tubo e microplaca. Os testes confirmatórios para a presença de D fraco incluíram o teste de (AGH) em tubo, gel e aderência de glóbulos vermelhos em fase sólida (F.S). Todas as amostras de DNA foram extraídas do sangue periférico e as variantes D foram classificadas utilizando diferentes ensaios moleculares. Resultados: As variantes de RHD identificadas por análise molecular incluíram os tipos D fracos 1, 2, 3, 11 e 38 e D parcial (DAR1.2, DAR1, DAR3.1, DAU0, DAU2, DAU4, DAU5, DAU6, DMH e DVII). O monoclonal Blend RUM-1/MS26 foi o melhor reagente anti-D utilizado para detectar o antigénio D na fase de LI em tubo, reagindo com 57% das variantes D com reactividade >2+, enquanto que o blend anti-D D175+ 415 foi o melhor monoclonal utilizado em microplacas, reagindo com 82% das variantes D. Os testes em F.S demonstraram forte reatividade (3-4+) com 100% das amostras testadas. Conclusão: Este estudo nos permitiu comparar e determinar a metodologia e os reagentes mais apropriados para identificar as variantes de D mais freqüentes. Nossos resultados mostram que mesmo usando métodos e regentes sensíveis para assegurar a correta caracterização do antígeno D, pelo menos 17% das amostras do doador necessitaram de um teste confirmatório de D para evitar a aloimunização em pacientes D negativosAbstract: D antigen is the most immunogenic antigen in the Rh blood group system , The D antigen is highly polymorphic and certain mutations and /or deletions lead to a weak phenotype defined by decreased density of antigen sites which require the use of indirect antiglobulin test (IAT) for detection. In this study we evaluated different technologies and clones of anti-D to propose the most appropriate serologic strategy to detect the largest numbers of D variants in blood donors. Methods:We selected 101 samples from Brazilian blood donors with different expressions of D in our donor routine. The tests were performed at immediate spin (IS) with eleven commercially available anti-D reagents in tube and microplate. D confirmatory tests for the presence of weak D included the (IAT) test in tube, gel and solid-phase red blood cell adherence (SPRCA). All DNA samples were extracted from peripheral blood and the D variants were classified using different molecular assays. Results: RHD variants identified by molecular analysis included weak D types 1, 2, 3, 11 and 38 and partial Ds (DAR1.2, DAR1, DAR3.1, DAU0, DAU2, DAU4, DAU5, DAU6, DMH and DVII).The RUM-1/MS26 monoclonal-monoclonal blended was the best anti-D reagent used to detect the D antigen at the IS phase in tube, reacting with 57% of the D variants with reactivity >2+, while the anti-D blend D175+415 was the best monoclonal (MoAb) used in microplate, reacting with 82% of the D variants. D confirmatory tests using SPRCA showed strong positive reactivity (3-4+) with 100% of the D variant samples tested.Conclusion:This study allowed us compare and determine the most appropriate methodology and reagents to identify the most frequent D variants. Our results show that even using sensitive methods and MoAbs to ensure the accurate assignment of D antigen, at least 17% of the donor samples need a confirmatory D test in order to avoid alloimmunization in D-negative patients ?MestradoClinica MedicaMestra em Ciência

Characterization of RHD alleles present in serologically RHD-negative donors determined by a sensitive microplate technique

Weak D phenotypes with very low antigen densities and DEL phenotype may not be detected in RhD typing routine and could be typed as D-negative, leading to D alloimmunization of D-negative recipients. The present study aimed to investigate the presence of RHD-positive genotypes in blood donors typed as D-negative by an automated system using the solid-phase methodology as a confirmatory test. Two screenings were performed in different selected donor populations. For the first screening, we selected 1403 blood donor samples typed as D-negative regardless of the CE status, and in the second screening, we selected 517 donor samples typed as D-negative C+ and/or E+. RhD typing was performed by microplate in an automated equipment (Neo-Immucor (R)), and the confirmatory test was performed by solid-phase technique using Capture R (R) technology. A multiplex PCR specific to RHD and RHD psi was performed in a pool of 6 DNA samples. Sequencing of RHD exons was performed in all RHD-positive samples, and a specific PCR was used to identify the D-CE(4-7)-D hybrid gene. No weak D type was found in either screening populations. Additionally, 353 (18 center dot 4%) D-negative samples presented previously reported non-functional RHD genes, 2 samples had a DEL allele, and 6 samples demonstrated new alleles, including one novel DEL allele. Our study identified six new RHD alleles and showed that the inclusion of a confirmatory test using serological methodology with high sensitivity can reduce the frequency of weak D samples typed as D-negative114886987

A new strategy to identify rare blood donors: Single polymerase chain reaction multiplex SNaPshot reaction for detection of 16 blood group alleles

Background. As an alternative to phenotyping, large-scale DNA-based assays, which are feasible for high-throughput donor red blood cell typing, were developed for determination of blood group polymorphisms. However, high-throughput genotyping platforms based on these technologies are still expensive and the inclusion of single nucleotide polymorphisms and analysis of the alleles depend on the manufacturer's determination. To overcome this limitation and in order to develop an assay to enable the screening of rare donors, we developed a SNaPshot assay for analysis of nine single nucleotide polymorphisms related to antigens that are difficult to assess using conventional serology. Materials and methods. The single polymerase chain reaction multiplex SNaPshot reaction was optimized to identify nine single nucleotide polymorphisms determining 16 alleles: KEL*3/KEL*4, KEL*6/KEL*7, DI*1/DI*2, DI*3/DI*4, YT*1/YT*2, CO*1/CO*2, DO*1/DO*2, DO*4, DO*5. We designed a single multiplex PCR with primers encompassing the blood group single nucleotide polymorphisms and performed an internal reaction with probe primers able to discriminate the alleles after fragment analysis. The SNaPshot assay was validated with 140 known alleles previously determined by PCR restriction fragment length polymorphism. Results. We were able to simultaneous detect nine single nucleotide polymorphisms defining 16 blood group alleles on an assay based on a multiplex PCR combined with a single base extension using genomic DNA. Discussion. This study demonstrates a robust genotyping strategy for conducting rare donor screening which can be applied in blood centers and could be an important tool for identifying antigen-negative donors and, therefore, for providing rare blood121s256s263sem informaçã