Implementing Prenatal Diagnosis Based on Cell-Free Fetal DNA: Accurate Identification of Factors Affecting Fetal DNA Yield

Angela N Barrett,Bernhard G Zimmermann,Darrell Wang,Andrew Holloway,Lyn S Chitty,Giuseppe Novelli

doi:10.1371/journal.pone.0025202

Abstract

ObjectiveCell-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.MethodsMicrofluidic 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.ResultsTotal cell-free DNA quantity increased significantly with time in samples stored in K3EDTA 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°C did not prevent these changes.ConclusionWhen samples can be processed within eight hours of blood draw, K3EDTA tubes can be used. Prolonged transfer times in K3EDTA 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.

Highlights

The presence of cell-free fetal DNA in the maternal circulation offers an alternative source of fetal genetic material for prenatal diagnosis [1]
When samples can be processed within eight hours of blood draw, K3EDTA tubes can be used
Prolonged transfer times in K3EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable

Summary

Introduction

The presence of cell-free fetal DNA in the maternal circulation offers an alternative source of fetal genetic material for prenatal diagnosis [1]. NIPD based on cell-free fetal DNA in situations where both parents carry a mutant allele for recessively-inherited monogenic disorders or for the diagnosis of fetal aneuploidy is more challenging and may depend on detection of small changes in relative proportions of alleles using methods such as digital PCR [13,14] or generation sequencing [15,16,17]. In this context, optimization of the proportion of cell-free fetal DNA yield may become critical. Further studies using blood taken from pregnant women showed a similar increase in total cell-free DNA over time, but using realtime PCR demonstrated that the absolute quantity of cell-free fetal DNA remained constant [19]

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Discussion

Conclusion