Cell-free fetal DNA (cffDNA) enrichment for non-invasive prenatal testing (NIPT): a comparison of molecular techniques
Date
2016Author
Subject
Metadata
Show full item recordAbstract
Prenatal assessment of fetal health is routinely offered throughout pregnancy to ensure that the most effective management can be provided to maintain fetal and maternal well-being. Currently, invasive testing is used for definitive diagnosis of fetal aneuploidy, which is associated with a 1% risk of iatrogenic fetal loss. Developing non-invasive prenatal testing (NIPT) is a key area of research and methods to increase the level of cell-free fetal DNA (cffDNA) within the maternal circulation have been discussed to improve accuracy of such tests. In this study, three strategies; co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR), inverse-PCR and Pippin Prep™ gel electrophoresis, were analysed to identify a novel approach to selectively enrich shorter cffDNA fragments from larger maternal cell-free DNA (cfDNA). The sensitivity of droplet digital PCR (ddPCR) against real-time PCR (qPCR) was compared for fetal sex and RHD genotyping. In addition RHD zygosity testing was carried out for non-maternal samples. Consequently, Pippin Prep™ gel electrophoresis was combined with ddPCR analysis for the NIPD of Down Syndrome (DS) in pseudo-maternal samples. The results revealed that the Pippin Prep™ gel electrophoresis enrichment approach successfully demonstrated 2-fold to 5-fold increases in the cffDNA fraction. However, further optimisation assays of COLD-PCR and inverse-PCR using actual maternal samples were required. The spike experiments for DS detection revealed that with the present assay IV overrepresentation of the chromosome 21 target could be significantly detected for samples with ≥15% ‘cffDNA fraction’. In conjunction with the Pippin Prep™ enrichment method, this would have enabled assessment of all 10 maternal samples. Alternatively, fetal sex and RHD genotyping results determined that ddPCR provides a more sensitive platform compared to qPCR approaches, particularly for samples that express low cffDNA fractions (<2%). The ddPCR platform also proved to be a rapid and accurate system for the determination of RHD zygosity. This study highlights that ddPCR could be used as opposed to qPCR for accurate determination of fetal sex and RHD status. While sequencing approaches currently provide the most sensitive platforms for NIPT of fetal aneuploidy, high costs (>£400) prevent universal application. The combination of cffDNA enrichment with ddPCR analysis could provide a cheaper and more widely available platform for NIPD. However, further large scale validation studies using actual maternal samples are required.
Collections
Publisher
Commissioning body
The following license files are associated with this item:
Related items
Showing items related by title, author, creator and subject.
-
Green-top Guideline 57: Reduced Fetal Movements
Fisher, Margaret; Whitworth, M; Heazell, A (Royal College of Obstetricians and Gynaecologists, 2011-02-01)The purpose of this guideline is to provide advice to guide clinicians, based on the best evidence where available, regarding the management of women presenting with reduced fetal movements (RFM) during pregnancy. This ... -
Impact of fetal or child loss on parents' perceptions of non-invasive prenatal diagnosis for autosomal recessive conditions
Pisnoli, L; O'Connor, A; Goldsmith, L; Jackson, L; Skirton, Heather (Elsevier BVScotland, 2016-03)OBJECTIVE: to explore parents' personal attitudes towards non-invasive prenatal diagnosis in the context of their own experiences caring for a child affected with a genetic condition or after the loss of a fetus, infant, ... -
Non Invasive Prenatal Diagnosis of Aneuploidy: Next Generation Sequencing or Fetal DNA Enrichment?
Avent, Neil; Webb, A; Madgett, TE; Miran, T; Sillence, K; Kaushik, N; Kiernan, Michele (Walter de Gruyter GmbHPoland, 2012-12-01)<jats:title>ABSTRACT</jats:title> <jats:p> Current invasive procedures [amniocentesis and chorionic villus sampling (CVS)] pose a risk to mother and fetus and such diagnostic procedures are available only to high risk ...