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
Fetal and Neonatal alloimmune thrombocytopenia (FNAIT) is a condition which could occur when pregnant women develop an alloimmunization against paternally inherited antigens of the fetal platelets. Approximately 80 % of FNAIT cases are caused by anti-HPA-1a, about 15 % by anti-HPA-5b and 5 % by other HPA antibodies. Only 2 % of the total population is HPA-1a negative (HPA-1b1b). The HPA-1a allele differs by one single nucleotide from HPA-1b allele, yet it represents around 27 % of total severe thrombocytopenias. HPA-1 was studied in serum cDNA from 12 volunteer pregnant women to determine their HPA-1 genotype by HRM (high resolution melting) PCR. When an homozygous HPA-1 gene was detected in a mother, a COLD HRM was performed to determine whether or not the fetal genotype differs from the mother's.The differences in the melting curve shapes allow us to accurately distinguish the three pregnants genotypes. The fetal heterozygous genotype of homozygous pregnant women was correctly detected by COLD PCR HRM in maternal serum. HPA-1 genotyping by HRM may be a useful aproach for genotyping all pregnant women in inexpensively. Moreover, when HPA-1 homozygosis was detected in a pregnant woman, fetal heterozygosis may be diagnosed by COLD HRM to select pregnancies for preventive monitoring.
Assuntos
Plaquetas/metabolismo , Troca Materno-Fetal/imunologia , Diagnóstico Pré-Natal/métodos , Trombocitopenia Neonatal Aloimune/imunologia , Antígenos de Plaquetas Humanas/sangue , Antígenos de Plaquetas Humanas/genética , Antígenos de Plaquetas Humanas/imunologia , Análise Custo-Benefício , DNA/sangue , DNA/genética , Feminino , Genótipo , Técnicas de Genotipagem/economia , Técnicas de Genotipagem/métodos , Humanos , Recém-Nascido , Integrina beta3 , Troca Materno-Fetal/genética , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , Gravidez , Reprodutibilidade dos Testes , Análise de Sequência de DNA/métodos , Trombocitopenia Neonatal Aloimune/sangue , Trombocitopenia Neonatal Aloimune/genéticaRESUMO
OBJECTIVES: Gene frequencies of human platelet antigens (HPA) determine the magnitude of platelet immunological disorders like neonatal alloimmune thrombocytopenia, platelet refractoriness and ease of availability of particular HPA-typed platelet donors in a given community. BACKGROUND: However, the pattern of HPA in Pakistani population is not known. AIM: The aim of present study was to determine the gene frequencies of HPA (HPA-1 to -5 and -15) in individuals belonging to major ethnic groups and castes of Pakistani population. MATERIALS AND METHODS: HPA genotyping was done in 593 individuals belonging to all ethnic groups of Pakistan, by polymerase chain reaction-sequence specific primers with detection on polyacrylamide electrophoresis. RESULTS: The gene frequencies of the 'a' and 'b' alleles of HPA-1 to -5 and -15 in Pakistanis were as follows: HPA-1a/b, 0.885/0.115; HPA-2a/b, 0.92/0.08; HPA-3a/b, 0.69/0.31; HPA-4a/b, 1/0; HPA-5a/b, 0.9/0.1; HPA-15a/b, 0.59/0.41. Except for significant difference regarding gene frequency of HPA-3 between Pathans and Sindhis, there was no significant difference of HPA-1 to -5 and -15 between major ethnic groups of Pakistan. The estimated mismatch probability regarding platelet antigens 1-5 and 15 in Pakistanis, after transfusion of random donor platelets, is from 14 to 37%. The expected incidence of neonatal alloimmune thrombocytopenia due to anti-HPA-1a in Pakistani pregnant females is < 1 of 1000 pregnancies and 8-12 of 1000 in case of anti-HPA-5b. Homozygosity of HPA-1b, -2b and -5b genotypes ranged from 1 to 2% in the Pakistani population, whereas homozygosity of HPA-3b and -15b was 11 and 18%. CONCLUSIONS: There is a need to establish donor registries typed for HPA in the transfusion centres of the country.