RESUMO
BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay. STUDY DESIGN AND METHODS: A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500. RESULTS: The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03. CONCLUSION: A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions.
Assuntos
Antígenos de Plaquetas Humanas/análise , Ensaios de Triagem em Larga Escala , Testes Sorológicos , Antígenos de Plaquetas Humanas/sangue , Antígenos de Plaquetas Humanas/genética , Estudos de Coortes , Análise Custo-Benefício , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Genótipo , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/métodos , Teste de Histocompatibilidade/economia , Teste de Histocompatibilidade/métodos , Humanos , Recém-Nascido , Integrina beta3 , Isoanticorpos/análise , Isoanticorpos/sangue , Países Baixos , Fenótipo , Valor Preditivo dos Testes , Gravidez , Sensibilidade e Especificidade , Testes Sorológicos/economia , Testes Sorológicos/métodos , Trombocitopenia Neonatal Aloimune/sangue , Trombocitopenia Neonatal Aloimune/diagnóstico , Trombocitopenia Neonatal Aloimune/genética , Trombocitopenia Neonatal Aloimune/imunologia , Fatores de TempoRESUMO
Most blood group antigens are defined by single nucleotide polymorphisms (SNPs). Highly accurate MALDI-TOF MS has proven its potential in SNP genotyping and was therefore chosen for blood donor oriented genotyping with high-throughput capability, e.g., 380 samples per day. The Select Module covers a total of 36 SNPs in two single-tube reactions, representative of 46 blood group and 4 human platelet antigens. Using this tool, confirmatory blood group typing for RhD, RhCE, Kell, Kidd, Duffy, MN, Ss, and selected rare antigens is performed on a routine basis.
Assuntos
Antígenos de Plaquetas Humanas/genética , Antígenos de Grupos Sanguíneos/genética , Técnicas de Genotipagem/métodos , Polimorfismo de Nucleotídeo Único , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Antígenos de Plaquetas Humanas/análise , Sequência de Bases , Antígenos de Grupos Sanguíneos/análise , DNA/análise , DNA/genética , Genótipo , Técnicas de Genotipagem/economia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economiaRESUMO
A simplified method for large-scale HPA-1a phenotyping of platelets was developed for use in an antenatal screening programme for fetomaternal alloimmune thrombocytopenia (FMAIT). The test was based on the MAIPA assay, which was modified for antigen-typing with a well-characterized anti-HPA-1a reagent. The resulting assay gave reliable results, was inexpensive and allowed testing of large batches using semiautomated equipment.
Assuntos
Antígenos de Plaquetas Humanas/análise , Testes Genéticos , Gravidez/sangue , Trombocitopenia/prevenção & controle , Anticorpos Monoclonais/imunologia , Antígenos de Plaquetas Humanas/genética , Bases de Dados Factuais , Feminino , Testes Genéticos/economia , Humanos , Técnicas Imunoenzimáticas , Integrina beta3 , Fenótipo , Gravidez/imunologia , Sorologia , Trombocitopenia/genética , Trombocitopenia/imunologiaRESUMO
Neonatal alloimmune thrombocytopenia (NAIT), which usually involves sensitization to P1A1 (HPA-1a), may have devastating complications for the fetus. These may be prevented by antenatal treatment of severe cases with either maternally administered high-dose gamma-globulin and/or repeated intrauterine platelet transfusions. Determination of the paternal platelet phenotype is useful for counseling parents who have had one or more affected pregnancies. This report of an unaffected pregnancy in a woman with a history of previous pregnancies complicated by NAIT illustrates the role of paternal and fetal platelet phenotyping in managing existing pregnancies at risk of NAIT.