Quantification of cell-free DNA in normal and complicated pregnancies: overcoming biological and technical issues.

Irina Manokhina,Maria S Peñaherrera,Wendy P Robinson,Tanjot K Singh,Maj Hulten

doi:10.1371/journal.pone.0101500

Irina Manokhina, Maria S Peñaherrera + Show 3 more

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https://doi.org/10.1371/journal.pone.0101500

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Abstract

The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal plasma provides a powerful tool for non-invasive diagnosis of fetal and obstetrical complications. Due to its placental origin, the specific epigenetic features of this DNA (commonly known as cell-free fetal DNA) can be utilized in creating universal ‘fetal’ markers in maternal plasma, thus overcoming the limitations of gender- or rhesus-specific ones. The goal of this study was to compare the performance of relevant approaches and assays evaluating the amount of cfDNA in maternal plasma throughout gestation (7.2–39.5 weeks). Two fetal- or placental- specific duplex assays (RPP30/SRY and RASSF1A/β-Actin) were applied using two technologies, real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). Both methods revealed similar performance parameters within the studied dynamic range. Data obtained using qPCR and ddPCR for these assays were positively correlated (total cfDNA (RPP30): R = 0.57, p = 0.001/placental cfDNA (SRY): R = 0.85, p<0.0001; placental cfDNA (RASSF1A): R = 0.75, p<0.0001). There was a significant correlation in SRY and RASSF1A results measured with qPCR (R = 0.68, p = 0.013) and ddPCR (R = 0.56, p = 0.039). Different approaches also gave comparable results with regard to the correlation of the placental cfDNA concentration with gestational age and pathological outcome. We conclude that ddPCR is a practical approach, adaptable to existing qPCR assays and well suited for analysis of cell-free DNA in plasma. However, it may need further optimization to surpass the performance of qPCR.

Highlights

The quantification and characterization of cell-free DNA has opened up new possibilities for prenatal diagnosis and screening [1], as well as for monitoring diseases such as cancer [2]
This study provides an assessment of the relevant approaches and assays for absolute fractional quantification of cell-free DNA (cfDNA) in maternal plasma
The measurement of cfDNA in maternal plasma broadens the possibilities for prenatal diagnosis and pregnancy monitoring, but poses specific technical and biological challenges unique to cell-free nucleic acids in biological fluids

Summary

Introduction

The quantification and characterization of cell-free DNA (cfDNA) has opened up new possibilities for prenatal diagnosis and screening [1], as well as for monitoring diseases such as cancer [2]. Placenta-derived cfDNA level is affected by gestational age and the health of the placenta; for example, aneuploidy or preeclampsia can alter trophoblast growth and death dynamics [13]. In conditions such as preeclampsia, both maternal and placental cfDNA increase, the absolute quantification of cfDNA, but not the ratio, may be predictive of disease onset. There are no generally accepted units of measure for cfDNA quantification; in the literature data are presented in genome equivalents per mL of plasma (GE/mL) [21,24,25,26,27] or its logged equivalent [27], median CT [28,29], percent value to total cfDNA [30,31] or multiple of median (MoM) [11]. This inconsistency complicates the data analysis, comparability and reproducibility of the tests

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