Serologic strategy in detecting RHD altered alleles in Brazilian blood donors

ABSTRACT Background: We evaluated different technological approaches and anti-D clones to propose the most appropriate serologic strategy in detecting 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 in immediate spin (IS) with eleven commercially available anti-D reagents in a tube and microplate. The D confirmatory tests for the presence of weak D included the indirect antiglobulin test (IAT) in a 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: The 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 monoclonal-monoclonal blend RUM-1/MS26 was the best anti-D reagent used in detecting the D antigen in the IS phase in a tube, reacting with 83.2% of the D variants, while the anti-D blend D175 + 415 was the best monoclonal antibody (MoAb) used in a microplate to minimize the need for an IAT, reacting with 83.2% of the D variants. The D confirmatory tests using SPRCA showed a reactivity (3 - 4+) with 100% of the D variant samples tested. Conclusion: Our results show that, even using sensitive methods and MoAbs to ensure the accurate assignment of the D antigen, at least 17% of our donor samples need a confirmatory D test in order to avoid alloimmunization in D-negative patients.

Serologic strategy in detecting RHD altered alleles in Brazilian blood donors.

BACKGROUND: We evaluated different technological approaches and anti-D clones to propose the most appropriate serologic strategy in detecting 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 in immediate spin (IS) with eleven commercially available anti-D reagents in a tube and microplate. The D confirmatory tests for the presence of weak D included the indirect antiglobulin test (IAT) in a 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: The 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 monoclonal-monoclonal blend RUM-1/MS26 was the best anti-D reagent used in detecting the D antigen in the IS phase in a tube, reacting with 83.2% of the D variants, while the anti-D blend D175 + 415 was the best monoclonal antibody (MoAb) used in a microplate to minimize the need for an IAT, reacting with 83.2% of the D variants. The D confirmatory tests using SPRCA showed a reactivity (3 - 4+) with 100% of the D variant samples tested. CONCLUSION: Our results show that, even using sensitive methods and MoAbs to ensure the accurate assignment of the D antigen, at least 17% of our donor samples need a confirmatory D test in order to avoid alloimmunization in D-negative patients.

Correlation among automated scores of agglutination, antigen density by flow cytometry and genetics of D variants.

BACKGROUND: Laboratory testing to identify the molecular basis of serologic weak D phenotypes is recommended to determine whether a pregnant woman or potential transfusion recipient should be managed as RhD-positive or RhD-negative. The variation in D antigen expression on RBCs, different potencies of anti-D typing reagents, lack of standardized test methods, and the subjectivity of interpreting agglutination reactions complicate the detection of D variants. We evaluated the correlation of agglutination scores by an automated immunoassay analyzer with D antigen densities determined by flow cytometry, and D variant types identified by molecular analysis. MATERIALS AND METHODS: We selected 273 blood donor samples with agglutination scores of less than 92 (4+), measured by an automated analyzer (NEO®, Immucor, Norcross, GA, USA). D antigen densities were measured by flow cytometry for 89 samples. Samples were classified as molecularly-determined weak D or partial D variants by multiplex PCR, PCR RFLP and DNA sequencing. RESULTS: All samples with a D antigen density ≥15% had an agglutination score >80 (4+). Agglutination scores for weak D types varied from 10 to 90. Agglutination scores for partial D antigens were graded with scores varying from 60 to 99. D antigen densities varied from 0.55% to 10.67% for weak Ds and 4.1% to 30.5% for partial Ds. DISCUSSION: Our results showed that score values follow a pattern among D variants that could be related to antigen density and to the RhD variant classification.

Novel RHAG allele encoding the Rh(null) phenotype in Brazil.

Rhnull is a rare phenotype characterized by the loss of Rh antigen expression. This phenotype can be related to several molecular backgrounds. In this study, we show a novel allele in a Brazilian pregnant woman encoding the Rhnull phenotype due to a change in RHAG exon2 c.310C>T, which leads to a premature stop codon (Gln104Stop).