Performance of non-invasive prenatal testing when fetal cell-free DNA is absent

Numerous studies have validated the accuracy of non-invasive prenatal testing (NIPT) using fetal cell-free DNA (cfDNA) to assess the risk of fetal aneuploidies early in pregnancy1, and we have used this technology in our practice since 2012 in both lowand high-risk women2. We are aware that several factors influence the fraction of fetal cfDNA present in maternal blood. Such factors include gestational age and maternal weight3, as well as methods of sample collection and shipping conditions that may lead to maternal cell hemolysis. Some commercial laboratories assert that the accuracy of cfDNA testing is influenced by the amount of fetal cfDNA relative to that of maternal cfDNA. In these laboratories that report fetal fraction, the performance claims for NIPT are based on testing that requires a minimal amount of fetal cfDNA to be present. We are also aware that some commercial laboratory providers assert that measurement of fetal cfDNA is unnecessary and that reliable results can be provided without prior knowledge of the amount of fetal cfDNA analyte in the sample. In order to assess the reliability of NIPT, blood samples from two 44-year-old non-pregnant women were drawn and submitted to five American commercial laboratories offering NIPT. The first sample was sent in September 2014 and the second in October 2014. We reported the gestational age of both pregnancies as 12 weeks on each requisition form, and did not inform any of the five laboratories that the women were in fact not pregnant. Each laboratory was paid out-of-pocket and no third-party was billed. The NIPT results provided to us by each laboratory are presented in Table 1. Two laboratories reported that there was insufficient fetal DNA present in the sample to provide a result. Three laboratories, two of which do not measure fetal