RESUMO
An automated magnetic capture hybridization (MCH) method for the extraction and enrichment of fetal RHD specific DNA fragments from maternal plasma was developed using plasma from 1000 D-negative pregnant women. A real time PCR protocol for RHD exon 7 was applied. MCH was compared with the QIAamp DSP Virus Kit (QIAamp) as a reference. Compared with the QIAamp method, the percentage of fetal DNA increased from 2.86% to 4.83% (p<0.05, n=8). The 95% detection limit of MCH was determined at 286 pg/ml (43 geg/ml) compared with 138 pg/ml (21 geq/ml) for the QIAamp DSP Virus Kit.
Assuntos
Tipagem e Reações Cruzadas Sanguíneas/métodos , DNA/isolamento & purificação , Troca Materno-Fetal , Sistema do Grupo Sanguíneo Rh-Hr/genética , DNA/análise , Sondas de DNA , Feminino , Humanos , Magnetismo , Técnicas de Amplificação de Ácido Nucleico , GravidezRESUMO
For fetal DNA extraction, in principle each DNA extraction method can be used; however, because most methods have been optimized for genomic DNA from leucocytes, we describe here the methods that have been optimized for the extraction of fetal DNA from maternal plasma and validated for this purpose in our laboratories. The use of the QIAamp DSP Virus kit (QIAGEN), the QIAamp DNA Blood Mini kit (QIAGEN), and the Magna Pure LC (Roche) is based on the kit components provided by the respective companies. However, we noticed that the yield of fetal DNA from maternal plasma can be increased when higher volumes are processed or some slight modifications of the protocols provided by the manufacturer are followed. Here, we also describe an in-house method that allows the specific capture of target molecules in an extremely low volume by using magnetic beads and magnetic tips. This method can be either performed by hand, or it can be adapted to a commercially available pipetting workstation.
Assuntos
DNA/sangue , Feto/metabolismo , Testes Genéticos , Diagnóstico Pré-Natal/métodos , DNA/isolamento & purificação , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Magnetismo , Troca Materno-Fetal , Gravidez , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Previous data indicate that a transfer of specific humoral and cellular immunity by way of allogeneic hematopoietic cell transplantation (HCT) should, in principle, be possible. METHODS: In the HCT setting with a follow-up of up to 55 months, we studied the transfer of hepatitis B virus (HBV) specific immunity from electively immunized donors into HLA compatible recipients suffering from chronic myeloid leukemia (CML). After excluding preexisting HBV specific immunity in donor-recipient pairs, 27 prospective donors were vaccinated against HBV. In addition, on an average of 22 months postHCT, 8 of the 19 recipients were immunized once for HBV. RESULTS: Donor vaccination resulted in detectable hepatitis B surface (HBs) antibodies in 85% of donors and specific cellular in vitro responses in 77%. Two weeks postHCT, 86 and 67% of the recipients displayed positive humoral and cellular HBV reactions, respectively, which then decreased. Afterwards, HBV immunity reappeared in 83% of the recipients without revaccination. Following a single vaccination in recipients, seven of eight displayed a typical memory response. An HBV specific response was already detectable 1 week after vaccination, approximately 1,300-fold (humoral) and 60-fold (cellular) higher than observed in the corresponding donors after a single immunization. CONCLUSIONS: The "spontaneous" recurrence of HBV immunity and the memory response in recipients give evidence for an elective immune transfer (e.g., for viral antigens) by way of allogeneic HCT.
