Since the first report in 1997, cell-free fetal DNA in the maternal circulation has become a primary target for noninvasive prenatal diagnosis (1). The detection of male-specific (Y chromosome) DNA sequences has been used for the assessment of X-linked disorders (2), the detection of unique gene sequences such as the RhD locus to determine fetomaternal blood group incompatibility (3), and the detection of dominantly inherited, paternally derived mutations for diagnoses of single gene disorders (1). The quantity of fetal DNA in maternal plasma or serum has been used as a marker of genetic disorders and complications of pregnancy, including common trisomies (4-7), preterm labor (8), and preeclampsia (9). Real-time PCR amplification of Y-chromosome sequences from the plasma or serum of women pregnant with male fetuses has been used in many studies as a model for the detection and quantification of fetal DNA. Y-chromosome DNA has been detected as early as 5 weeks of gestation (10, 11), before the time period in which invasive testing is typically performed. Thus, early detection of fetal DNA could have a profound effect on the way pregnancies are managed. Some investigators have reported 95% sensitivity and specificity for real-time PCR (12,13). However, these data are typically generated by a single laboratory under specific conditions and often include data from a small number of patients. In addition, confounders that could impact routine clinical application may be undetected in smaller studies. Indeed, it has been shown that methods of processing can drastically affect the amount of DNA detected by real-time PCR (14). For PCR assays to become part of standard prenatal care, their accuracy and reproducibility must be improved and the underlying variables that affect performance must be better understood.