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Letters to the Editor |
1 Division of Genetics, Department of Pediatrics 2
Institute for Clinical Research, and Health Policy Studies, Tufts-New England Medical Center, Boston, MA
3 Department of Pathology, Women and Infants Hospital, Providence, RI
aAddress correspondence to this author at: Division of Genetics, Department of Pediatrics, Tufts-New England Medical Center, 750 Washington St., Box 394, Boston, MA 02111. Fax 617-636-1469; e-mail kjohnson{at}tufts-nemc.org.
To the Editor:
Cell-free fetal nucleic acids (cffDNA) are present in maternal plasma and serum (1), but amniotic fluid (AF) also provides an attractive source of cffDNA. The concentration of cffDNA is 100- to 200-fold higher in AF than in maternal plasma/serum (2), but low yields of cffDNA compromise testing by techniques such as genomic microarrays, which require a minimum of 100 ng of DNA (3).
For protocol optimization, we used 5 large-volume AF supernatant samples from patients who had undergone therapeutic amnioreduction for twin-twin transfusion syndrome. After optimization, we compared the DNA yield of the old and new protocols for freshly discarded AF supernatant samples from 29 euploid singleton pregnancies. Approval for this study was obtained from the Institutional Review Boards of Tufts-New England Medical Center and Women and Infant’s Hospital. The median gestational age at amniocentesis was 16.9 weeks (25th–75th percentiles, 16.4–18.1 weeks).
We changed our original method (3) in the following 3 ways: we increased the vacuum extraction pressure to 800 mbar; we replaced the volume-overloaded mini spin columns with maxi spin columns (Qiagen) to allow for larger starting volumes; and we replaced the QiaAmp Lysis (AL) buffer with proprietary QiaAmp Viral Lysis (AVL) buffer (Qiagen), which has a high chaotropic salt concentration, on the basis of the similarities of AF and urine, a body fluid for which AVL buffer is recommended.
Quantitative PCR analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locus was performed in triplicate for each sample (4), with the mean result of the 3 reactions used for further calculations. The results were expressed as genome equivalents (GE) per milliliter, using a conversion factor of 6.6 pg of DNA per cell (5).
For large-volume AF samples, replacing AL with AVL buffer (using mini columns with high vacuum) led to a mean (SD) DNA yield of 1470 (456) GE/mL, and replacing mini with maxi columns (using AL buffer) led to a mean DNA yield of 1564 (623) GE/mL. Finally, substituting AL with AVL buffer and replacing mini with maxi columns led to a mean DNA yield of 1972 (786) GE/mL. DNA extraction with phenol, chloroform, and isoamyl alcohol (6) did not further improve the yield from 1 large-volume AF sample.
For euploid singleton pregnancies (n = 29), the median amount of GAPDH DNA extracted from 10 mL of AF with the new protocol was 1700 GE/mL (25th–75th percentiles, 1071–4938 GE/mL) compared with 246 (93–524) GE/mL obtained with the original protocol (3) (P <0.0001, Wilcoxon signed-rank test). The proportion of samples that had a sufficient yield of extracted DNA for subsequent chromosome microarray analysis (i.e., 
100 ng) also increased compared with the original protocol, from 39% (28 of 72) (3) to 86% (25 of 29; P <0.0001, 
2 test).
The new protocol allowed extraction of cffDNA from 
10 samples in less than 3 h. The replacement of AL with AVL buffer eliminated the need for a heating bath during the lysis step, and fewer overall steps are involved in the protocol, which may reduce the potential for contamination. The cost of reagents and supplies for cffDNA extraction from a 10-mL AF supernatant sample is 
10-fold higher for the new protocol than for the original protocol (costs of $39 and $4, respectively).
An advantage of using AF supernatant is its availability without interfering with current standards of care or compromising fetal health. Another is the ability to freeze the supernatant sample at –80 °C without risking significant degradation of DNA over time (7). For research applications, the development of an optimized protocol could allow further investigation of the origin and kinetics of cffDNA. Finally, placental abnormalities and pregnancyassociated disorders may affect cffDNA concentrations in maternal serum (8)(9), whereas fetal disorders and contact between fetal organs (such as lungs, kidneys, and the gastrointestinal system) and AF may affect cffDNA concentrations in AF.
Acknowledgments
This work was supported by NIH Grant R01HD42053 and the Swiss National Fund (PBBSB-108590). We thank the staff of the Tufts-New England Medical Center Cytogenetics laboratory, specifically Sarah Beam, Tara Bond, Karen Krajewski, and Dyanna Lincoln, for their generous support in obtaining AF samples.
References
The following articles in journals at HighWire Press have cited this article:
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  O. Lapaire, D. W. Bianchi, I. Peter, B. O'Brien, H. Stroh, J. M. Cowan, U. Tantravahi, and K. L. Johnson Cell-Free Fetal DNA in Amniotic Fluid: Unique Fragmentation Signatures in Euploid and Aneuploid Fetuses Clin. Chem., March 1, 2007; 53(3): 405 - 411. [Abstract] [Full Text] [PDF]
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