RESUMEN
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.
Asunto(s)
ADN/sangre , Feto/metabolismo , Pruebas Genéticas , Diagnóstico Prenatal/métodos , ADN/aislamiento & purificación , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Magnetismo , Intercambio Materno-Fetal , Embarazo , Juego de Reactivos para Diagnóstico , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: To determine the accuracy of non-invasive fetal testing for the RHD gene in week 27 of pregnancy as part of an antenatal screening programme to restrict anti-D immunoglobulin use to women carrying a child positive for RHD DESIGN: Prospectively monitoring of fetal RHD testing accuracy compared with serological cord blood typing on introduction of the test. Fetal RHD testing was performed with a duplex real time quantitative polymerase chain reaction, with cell-free fetal DNA isolated from 1 mL of maternal plasma The study period was between 4 July 2011 and 7 October 2012. The proportion of women participating in screening was determined. SETTING: Nationwide screening programme, the Netherlands. Tests are performed in a centralised setting. PARTICIPANTS: 25 789 RhD negative pregnant women. MAIN OUTCOME MEASURES: Sensitivity, specificity, false negative rate, and false positive rate of fetal RHD testing compared with serological cord blood typing; proportion of technical failures; and compliance to the screening programme. RESULTS: A fetal RHD test result and serological cord blood result were available for 25 789 pregnancies. Sensitivity for detection of fetal RHD was 99.94% (95% confidence interval 99.89% to 99.97%) and specificity was 97.74% (97.43% to 98.02%). Nine false negative results for fetal RHD testing were registered (0.03%, 95% confidence interval 0.01% to 0.06%). In two cases these were due to technical failures. False positive fetal RHD testing results were registered for 225 samples (0.87%, 0.76% to 0.99%). Weak RhD expression was shown in 22 of these cases, justifying anti-D immunoglobulin use. The negative and positive predictive values were 99.91% (95% confidence interval 99.82% to 99.95%) and 98.60% (98.40% to 98.77%), respectively. More than 98% of the women participated in the screening programme. CONCLUSIONS: Fetal RHD testing in week 27 of pregnancy as part of a national antenatal screening programme is highly reliable and can be used to target both antenatal and postnatal anti-D immunoglobulin use.
Asunto(s)
Diagnóstico Prenatal , Isoinmunización Rh/diagnóstico , Sistema del Grupo Sanguíneo Rh-Hr/genética , ADN/aislamiento & purificación , Femenino , Sangre Fetal/química , Humanos , Países Bajos , Valor Predictivo de las Pruebas , Embarazo , Complicaciones Hematológicas del Embarazo/sangre , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Isoinmunización Rh/genética , Isoinmunización Rh/terapia , Globulina Inmune rho(D)/administración & dosificación , Globulina Inmune rho(D)/genética , Sensibilidad y EspecificidadAsunto(s)
Células Sanguíneas/metabolismo , Ácidos Nucleicos Libres de Células/metabolismo , Inflamación/patología , Tejido Parenquimatoso/metabolismo , Animales , Voluntarios Sanos , Humanos , Cinética , Lipopolisacáridos , Masculino , Ratones , Neutrófilos/metabolismo , Nucleosomas/metabolismo , Tejido Parenquimatoso/citología , Sepsis/sangre , Índice de Severidad de la EnfermedadRESUMEN
The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10(-15)). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.
Asunto(s)
Antígenos de Grupos Sanguíneos/genética , Membrana Eritrocítica/metabolismo , Eritrocitos/inmunología , Eliminación de Gen , Homocigoto , Proteínas de la Membrana/genética , Sitios de Carácter Cuantitativo , Alelos , Animales , Biomarcadores/metabolismo , Antígenos de Grupos Sanguíneos/inmunología , Antígenos de Grupos Sanguíneos/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Eritrocitos/metabolismo , Eritrocitos/patología , Exoma/genética , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Isoanticuerpos/inmunología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Embarazo , Pez Cebra/genéticaRESUMEN
OBJECTIVE: Cell free foetal DNA (cff DNA) extracted from maternal plasma is now recognized as a potential source for prenatal diagnosis but the methodology is currently not well standardized. To evaluate different manual and automated DNA extraction methods with a view to developing standards, an International Workshop was performed. METHODS: Three plasma pools from RhD-negative pregnant women, a DNA standard, real-time-PCR protocol, primers and probes for RHD were sent to 12 laboratories and also to one company (Qiagen, Hilden, Germany). In pre-tests, pool 3 showed a low cff DNA concentration, pool 1 showed a higher concentration and pool 2 an intermediate concentration. RESULTS: The QIAamp DSP Virus Kit, the High Pure PCR Template Preparation Kit, an in-house protocol using the QIAamp DNA Blood Mini Kit, the CST genomic DNA purification kit, the Magna Pure LC, the MDx, the M48, the EZ1 and an in-house protocol using magnetic beads for manual and automated extraction were the methods that were able to reliably detect foetal RHD. The best results were obtained with the QIAamp DSP Virus Kit. The QIAamp DNA Blood Mini Kit showed very comparable results in laboratories that followed the manufacturer's protocol and started with > or = 500 microL plasma. One participant using the QIAamp DNA Blood Midi Kit failed to detect reliably RHD in pool 3. CONCLUSIONS: This workshop initiated a standardization process for extraction of cff DNA in maternal plasma. The highest yield was obtained by the QIAamp DSP Virus Kit, a result that will be evaluated in more detail in future studies.
Asunto(s)
ADN/sangre , ADN/aislamiento & purificación , Feto , Pruebas Genéticas/métodos , Madres , Automatización , Femenino , Feto/metabolismo , Pruebas Genéticas/normas , Humanos , Relaciones Materno-Fetales , Reacción en Cadena de la Polimerasa/métodos , Embarazo , Diagnóstico Prenatal/métodos , Diagnóstico Prenatal/normasRESUMEN
BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma led to the development of assays to predict the fetal D status with RHD-specific sequences. Few assays are designed in such a way that the fetus can be typed in RHDpsi mothers and that RHDpsi fetuses are correctly typed. Owing to the limited knowledge about the mechanism responsible for the presence of fetal DNA in maternal plasma, precautions in developing prenatal genotyping strategies must be made. STUDY DESIGN AND METHODS: Real-time quantitative (RQ)-polymerase chain reaction (PCR) assays were developed for prenatal diagnostic use with cell-free fetal DNA from maternal plasma. An RQ-PCR assay on RHD exon 5 (amplicon 361 bp), negative on RHDpsi, was developed with genomic DNA and evaluated with cell-free fetal DNA. A previously published RHD exon 5 RQ-PCR (amplicon 82 bp) was duplexed with an in-house developed RHD exon 7 RQ-PCR and evaluated with cell-free fetal DNA from pregnant D-RHDpsi+ women. RESULTS: The RHD exon 5 361 bp assay showed on cell-free plasma DNA from D- women carrying a D+ fetus, low amplification levels, resulting in high Ct values and false-negative results. Owing to fragmentation of cell-free plasma DNA, too few DNA stretches of sufficient length (> 360 bp) are present. The RHD exon 5 82 bp and exon 7 RQ-PCR duplex was evaluated with RHDpsi+ cell-free plasma DNA and showed complete specificity and maximal sensitivity. CONCLUSION: Assays designed for prenatal genotyping should be developed and evaluated on cell-free plasma DNA. Prenatal RHD typing is accurate with the RHD exon 5 82 bp and exon 7 duplex strategy.