Detecting fetal DNA from dried maternal blood spots: another step towards broad scale non-invasive prenatal genetic screening and feasible testing.

Both intact fetal cells and cell-free fetal DNA are present in the maternal circulation and have been used for non-invasive prenatal genetic diagnosis. However, broad clinical application awaits development of robust methods for collecting, transporting and enriching maternal blood samples to recover rare fetal cells. To circumvent this impediment, we have devised a reliable method of fetal DNA detection using dried maternal blood spots and real-time polymerase chain reaction. Fetal Y-specific (DYS1) sequences were detected in all 19 (100%) maternal blood specimens from women carrying male fetuses, in genome equivalents of 4.20-24.68 per ml of blood; the ubiquitous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, reflecting both maternal and fetal DNA, concurrently showed 43,684 to 680,357 genome equivalents per ml of blood. The results demonstrate that fetal DNA detection using dried maternal blood spots is highly feasible and easily adaptable for population screening.

Interlaboratory comparison of fetal male DNA detection from common maternal plasma samples by real-time PCR.

BACKGROUND: Analysis of fetal DNA from maternal plasma by PCR offers great potential for noninvasive prenatal genetic diagnosis. To further evaluate this potential, we developed and validated a standard protocol to determine whether fetal DNA sequences could be reproducibly amplified and measured across multiple laboratories in a common set of specimens. METHODS: Each of five participating centers in a National Institute of Child Health and Human Development consortium collected 20 mL of peripheral blood from 20 pregnant women between 10 and 20 weeks of gestation. The plasma fraction was separated according to a common protocol, divided, and frozen in five aliquots. One aliquot was shipped to each participating laboratory, where DNA was extracted according to a standard protocol. All plasma samples (n = 100) were then analyzed blindly for the presence and quantity of total DNA (GAPDH) and male fetal DNA (SRY) by real-time PCR. Genomic DNA was isolated from female and male cells at one center, quantified, and shipped to the others to serve as calibrators for GAPDH and SRY, respectively. RESULTS: The amplification of known quantities of DNA was consistent among all centers. The mean quantity of male DNA amplified from maternal plasma when the fetus was male ranged from 51 to 228 genome equivalents (GE)/mL. Qualitative concordance was found overall among centers. The sensitivity of the assay for detection of male DNA when the fetus was male varied from 31% to 97% among centers. Specificity was more consistent (93-100%) with only four false-positive results obtained across the entire study. CONCLUSIONS: All centers were able to consistently amplify frozen and shipped DNA. The PCR procedure used here is reliable and reproducible. Centers that extracted and amplified more DNA per milliliter of maternal plasma had superior sensitivities of Y chromosome sequence detection. The specificity of the assay was more consistent among centers. A robust and thoroughly optimized protocol for the extraction of DNA from maternal plasma is needed to make testing of fetal DNA in maternal plasma a clinically relevant analytical tool.