In researching methods to increase the proportion of fetal DNA recovered from maternal blood, and developing non-invasive methods for prenatal diagnosis, the main objective should be to contribute practical solutions to real-world problems.
Blood samples must be routinely transported from laboratories or physicians' offices to a testing facility and then processed, and there is great potential for maternal cell lysis because of mechanical shearing and degradation resulting from shipping, chemical processing, and centrifugation. Our formaldehyde method was developed to address this real-world problem. Addition of formaldehyde to maternal blood samples significantly increases the proportion of fetal DNA recovered from the plasma.1
Benachi and colleagues3
confirmed our results, reporting a range of 5·6—96% fetal DNA and a mean of 36·8% fetal DNA recovered from maternal plasma. We are perplexed by the apparent inability of others to practise this technique.
Fetal genotypes were confirmed by genotyping parental genomic DNA. Paternal genotyping was used for reference purposes to reduce the number of single-nucleotide polymorphisms (SNPs) analysed in the plasma, and is not required for quantitative analysis of fetal DNA. We compared our results with amniocentesis or newborn reports from the clinical sites. Our aim is to develop a completely non-invasive test not reliant on analysis of invasively obtained samples. Presentation of methods as “non-invasive” when they clearly rely on analysis of invasively obtained tissues is misleading.4
Sinuhe Hahn and colleagues state that our approach “is inferior to that attained in a report in which cell-free fetal mRNA was used.” If a comparison is to be made with other methods, it should at least be done accurately. We sequentially analysed 60 samples, none of which were excluded from our statistical analysis.2
In the paper cited in comparison as superior, Lo and colleagues collected and analysed 119 samples; however, 52 (44%) did not have a heterozygous phenotype at the locus of interest, and were removed from the dataset before statistical analysis. Lo and colleagues then reported successful identification of 55 of 57 normal samples and nine of ten trisomy 21 samples and estimated specificity and sensitivity based on 67 rather than the 119 samples collected, artificially improving their results. They fail to acknowledge that their methods were only diagnostic for 56% of the samples they collected.
As discussed in our paper, it is evident that the number of SNPs visible in the plasma often, although not consistently, relates to the proportion of fetal DNA in the sample. An additional practical benefit of this method is its ability to be done in the first trimester, allowing for analysis of a follow-up sample should a low number of SNPs be identified in the initial sample. Furthermore, unlike methods based on quantification of one or few SNPs on one or few chromosomes,4
the approach we describe is unlimited in terms of the number of reference chromosomes and number of SNPs that could be added to the analysis.
RD is the founder, chief executive officer, and chairman of the board of directors of Ravgen Inc, and a stockholder of that company. XG and SE are employed by, and have options to purchase stock in, Ravgen. MD is an unpaid member of Ravgen's advisory board, and has options to purchase stock. PB is a member of the board of directors of Ravgen and has options to purchase stock. Ravgen Inc has been issued patents and has multiple patent applications pending for the methods described in this letter.