Barrett, AN;
Zimmermann, BG;
Wang, D;
Holloway, A;
Chitty, LS;
(2011)
Implementing Prenatal Diagnosis Based on Cell-Free Fetal DNA: Accurate Identification of Factors Affecting Fetal DNA Yield.
PLOS ONE
, 6
(10)
, Article e25202. 10.1371/journal.pone.0025202.
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Abstract
Objective: Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used.Methods: Microfluidic digital PCR was performed to precisely quantify male (fetal) DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA). Real-time qPCR was used to assay for the presence of male SRY signal in samples.Results: Total cell-free DNA quantity increased significantly with time in samples stored in K(3)EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4 degrees C did not prevent these changes.Conclusion: When samples can be processed within eight hours of blood draw, K(3)EDTA tubes can be used. Prolonged transfer times in K(3)EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests.
| Type: | Article |
|---|---|
| Title: | Implementing Prenatal Diagnosis Based on Cell-Free Fetal DNA: Accurate Identification of Factors Affecting Fetal DNA Yield |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1371/journal.pone.0025202 |
| Publisher version: | http://dx.doi.org/10.1371/journal.pone.0025202 |
| Language: | English |
| Additional information: | © 2011 Barrett et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This article presents independent research commissioned by the National Institute for Health Research (NIHR) under the Programme Grants for Applied Research programme (the “RAPID” project). The authors acknowledge the support from the European Commission (Framework 6) Special Non-Invasive Advances in Fetal and Neonatal Evaluation (SAFE) Network of Excellence (LSHCT-2004-503241), the NIHR Programme Grants for Applied Research scheme and the NIHR Central and East London Comprehensive Local Research Network. LSC receives some funding from the NIHR Biomedical Research Centre for Paediatric/Child Health at Great Ormond Street Hospital NHS Trust and UCL Institute of Child Health and the Great Ormond Street Hospital Children's Charity. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. |
| Keywords: | MATERNAL PLASMA, FORMALDEHYDE TREATMENT, BLOOD, PROPORTION, IMPACT, SERUM, SIZE, DISEASES, SAMPLES, PCR |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Genetics and Genomic Medicine Dept |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1348110 |
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