Quantification of Cell-Free DNA in Normal and Complicated Pregnancies: Overcoming Biological and Technical Issues

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.

Advances in the Diagnosis of Human Schistosomiasis

“…In a recent advance in PCR technology, droplet digital PCR (dd PCR) was developed and has proven to be more sensitive and precise than qPCR (211)(212)(213). dd PCR has been used successfully for the detection of cellfree DNA and in the diagnosis of infections and other clinical conditions (211,214,215).…”

Cell-Free Placental DNA in Maternal Plasma in Relation to Placental Health and Function

“…The supernatant (plasma) was transferred to a new tube and centrifuged for 10 min at 16,000 g at 4 ° C. This second supernatant was then transferred to a new tube and stored at -80 ° C until DNA extraction. Cell-free DNA was extracted using the DNeasy Blood and Tissue Kit or QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, Calif., USA) as described before [24] .…”

“…The standard curve was obtained using a term placental DNA sample with known molecular load. Primer sequences and qPCR conditions are described in Manokhina et al [24] .…”

Noninvasive nucleic acid–based approaches to monitor placental health and predict pregnancy-related complications

“…43 However, this relatively new approach still needs to be validated for routine laboratory use. 44,45 Highthroughput techniques provide replete data that are useful for the elucidation of disease-specific changes in a sample genome (DNA), methylome (DNA methylation [DNAm] profile), or transcriptome (mRNA/miRNA profile).…”

“…Whereas it represents approximately 10% of total cell-free (cf) DNA, 50 its overall concentration is relatively low (mean approximately 100 genome equivalents per milliliter of plasma). 44 Also, interindividual variation in plasma cfp-DNA levels is quite high and is likely influenced by a number of factors (eg, ethnicity). 51 Abnormal levels of cfp-DNA may reflect active shedding of trophoblast particles or gradual breakdown of the placental barrier after the 32nd week of pregnancy, independently of pathology.…”