Using Size Selection to Enrich Fetal Fraction, Enabling Extended Blood Storage in EDTA tubes
Prenatal screening for Down’s Syndrome and other chromosomal imbalances by the analysis of a blood sample has been available since the 1980s. The original methods utilised maternal pregnancy markers, such as alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG), which lacked specificity (too many false positives) and sensitivity (too many false negatives).1 In 2011, the field was revolutionised by the introduction of noninvasive prenatal testing (NIPT), which directly measures the presence of placental-derived DNA, representing the fetus, circulating in the mother’s blood.2 This method is used today to screen pregnancies, minimising the number of people who needlessly undergo invasive testing, which has a higher risk of spontaneous miscarriage and has greatly improved sensitivity and specificity over the maternal markers.3,4
Cell-free DNA and Non-invasive Prenatal Testing
The technology we know today as NIPT was made possible by the first discovery of circulating cell-free fetal DNA (cffDNA) in the mother’s blood by Dennis Lo in 1997.5,6 The proportion of cffDNA in the mother’s blood that is derived from the fetus is referred to as the fetal fraction. It was immediately apparent that this had the potential to open the door to improved prenatal screening; however, at that time, no one could identify a method to analyse the fetal DNA amongst the maternal DNA circulating in the blood because of the relatively low fetal fraction, typically around 10% of cell-free (cfDNA) is cffDNA at 12 weeks gestation.
The insight that solved this problem was made, again, by Dennis Lo in 2008, who realised that it wasn’t necessary to separate the fetal and maternal DNA – instead, he developed a method based on counting chromosomal markers.7 Down’s Syndrome is caused by an extra copy of chromosome 21; therefore, a woman pregnant with a fetus affected by Down’s Syndrome will show a small increase in the amount of chromosome-21 DNA in circulation. Chromosome 21 represents about 2.1% of the human genome but in a pregnancy affected by Down’s Syndrome, this is increased to around 2.2% (depending on the fetal fraction). If it was feasible to measure this small but significant increase, then it would allow prenatal screening of trisomy 21. Technical advances in DNA sequencing that allowed the analysis and chromosomal mapping of millions of cffDNA fragments made it possible to accurately count and calculate the proportion of chromosome-21-derived DNA. A proportion of 2.1% was good evidence of an unaffected pregnancy; a higher proportion indicated the presence of trisomy 21. This insight led to the establishment of NIPT as we know it today, meaning fetal trisomy can be screened for from a maternal blood sample without the need for an invasive procedure which carries a risk of spontaneous miscarriage of healthy pregnancies.