RESUMO
BACKGROUND: Genetic diversity in the RH genes among sickle cell disease (SCD) patients is well described but not yet extensively explored in populations of racially diverse origin. Transfusion support is complicated in patients who develop unexpected Rh antibodies. Our goal was to describe RH variation in a large cohort of Brazilian SCD patients exhibiting unexpected Rh antibodies (antibodies against RH antigens to which the patient is phenotypically positive) and to evaluate the impact of using the patient's RH genotype to guide transfusion support. STUDY DESIGN AND METHODS: Patients within the Recipient Epidemiology and Evaluation Donor Study (REDS)-III Brazil SCD cohort with unexpected Rh antibodies were selected for study. RHD and RHCE exons and flanking introns were sequenced by targeted next-generation sequencing. RESULTS: Fifty-four patients with 64 unexplained Rh antibodies were studied. The majority could not be definitively classified as auto- or alloantibodies using serologic methods. The most common altered RH were RHD*DIIIa and RHD*DAR (RHD locus) and RHCE*ce48C, RHCE*ce733G, and RHCE*ceS (RHCE locus). In 53.1% of the cases (34/64), patients demonstrated only conventional alleles encoding the target antigen: five of 12 anti-D (41.7%), 10 of 12 anti-C (83.3%), 18 of 38 anti-e (47.4%), and one of one anti-E (100%). CONCLUSION: RHD variation in this SCD cohort differs from that reported for African Americans, with increased prevalence of RHD*DAR and underrepresentation of the DAU cluster. Many unexplained Rh antibodies were found in patients with conventional RH allele(s) only. RH genotyping was useful to guide transfusion to determine which patients could potentially benefit from receiving RH genotyped donor units.
Assuntos
Alelos , Transfusão de Sangue , Genótipo , Isoanticorpos/sangue , Sistema do Grupo Sanguíneo Rh-Hr , Anemia Falciforme/sangue , Anemia Falciforme/genética , Anemia Falciforme/terapia , Brasil , Feminino , Humanos , Masculino , Sistema do Grupo Sanguíneo Rh-Hr/sangue , Sistema do Grupo Sanguíneo Rh-Hr/genéticaAssuntos
Transfusão de Sangue , Técnicas de Genotipagem , Hematologia , Técnicas Imunológicas , Alergia e Imunologia/educação , Antígenos de Grupos Sanguíneos/análise , Transfusão de Sangue/métodos , Doenças Transmissíveis/sangue , Doenças Transmissíveis/genética , Doenças Transmissíveis/imunologia , Doenças Transmissíveis/terapia , Eritrócitos/imunologia , Eritrócitos/metabolismo , Técnicas de Genotipagem/métodos , Hematologia/educação , Hematologia/métodos , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/terapia , Humanos , Técnicas Imunológicas/métodos , Philadelphia , Sociedades MédicasRESUMO
A tecnologia dos eritrócitos magnetizados (E.M.®Technology) é uma grande inovação nos ensaios imunoematológicos. Os testes são realizados manualmente na estação detrabalho Freelys®Nano ou no equipamento automatizado Qwalys® (Diagast, Loos, France). O método utiliza hemácias magnetizadas e uma placa magnética substitui a centrifugação. As microplacas para a classificação sangüínea e fenotipagem Rh e K contêm anti-soros monoclonais IgM. As microplacas destinadas a pesquisa de anticorpos irregulares (PAI) e prova cruzada (PC) contêm antiglobulina humana (AGH) monoclonalmurina: anti-IgG na PAI e na PC anti-IgG + anti-IgM de baixo título para detectar anticorpos ABO. Na PAI e na PC, as reações acontecem na camada contendo AGH (imunocaptura) e visa detectar anticorpos IgG. A utilização de um meio de alta densidade possibilita a utilização da AGH sem lavagens prévias. Na avaliação da E.M.®Technology na classificação sangüínea, Fenotipagem, PAI e PC, na estação Freelys®Nano, foram utilizadas amostras de pacientes, doadores, sangue de cordão, hemácias de pacientes com teste de Coombs direto positivo e concentrados de hemácias. Todas as amostras foram paralelamente testadas em gel-teste (Diamed e Grifols). A concordância entre a E.M.®Technology e o gel-teste foi: 100% na classificação ABO e RhD e na fenotipagem Rh/ K, 94,6% na PAI e 92,3% na PC. A sensibilidade da E.M.®Technology na detecção de anticorpos IgG foi 95,5% em ambos os métodos. A E.M.®Technology mostrou um bom desempenho nos testes efetuados.
The erythrocytes magnetized technology (E.M.® Technology) is a great innovation in the field of blood banking. The tests can be performed manually on a Freelys® Nano workstation or on thefully-automated system QWALYS® (Diagast, Loos, France). This method does not require centrifugation steps thanks to the use of magnetic red blood cells and a magnetic plate. For blood grouping and Rh/K phenotyping, the microplate contains monoclonal IgMantibodies. The wells of the microplate to perform antibody screening and cross-matching are coated with murine monoclonal anti-human globulin: anti-IgG for antibody screening and anti-IgG + anti-IgM for cross-matching. The E.M.® Technology is only able to detectIgG antibodies. A high-density medium layer avoids the necessity of rinsing before the antiglobulin reagent. To evaluate the performance of E.M.® Technology on a Freelys® Nano work station for blood grouping, Rh/K phenotyping, antibody screening and crossmatching, we selected samples from patients, blood donors, blood cords, red blood cells from patients with positive direct antiglobulin test (DAT) and red blood cells from storage blood bags. In parallel, the tests were performed using the gel-technique (Diamed and Grifols). The concordance between E.M.® Technology and gel-testwas 100% for blood grouping and Rh/K phenotyping, 94.6% for antibody screening and 92.3% for cross-matching. The sensitivity to detect IgG antibodies was 95.5% in both methods. The E.M.® Technology on a Freelys® Nano workstation provided good resultsin all the tests performed.