Background: The cell free fetal DNA (cffDNA) present in plasma of pregnant women represents an important alternative source of DNA for non-invasive prenatal diagnosis. Due to the low quantity and increased fragmentation of cffDNA, the choice of DNA extraction method is a crucial step for downstream analyses.
Methods: In our study, the three spin column-based kits for isolation of cffDNA [DNA Blood Mini Kit (DBM), DSP Virus Kit (DSP) and Circulating Nucleic Acid (CNA) Kit] were compared. Original and optimized protocol were used in comparison and applied in the two phases of the study.
Results: A statistically significant difference in performance of the kits was determined based on the comparison of genomic equivalents per mL (GEq/mL) values (p<0.0001). The GEq/mL of isolated DNA was significantly higher using CNA and DSP Kits than DBM Kit. The CNA Kit and DSP Kit did not significantly differ in the GEq/mL values, although all tested samples isolated with CNA Kit showed higher values.
Conclusions: According to our results the commonly used DBM Kit could be successfully replaced with CNA or DSP Kits. The replacement could be beneficial in qualitative as well quantitative tests (e.g., gender determination, aneuploidy detection) when the isolation yield limits subsequent analyses. However, there is an important decision to be made when switching DBM Kit for DSP or CNA Kits. The price of DBM Kit is two and six times lower than DSP and CNA Kits, respectively.
Mujezinovic F, Alfirevic Z. Procedure-related complications of amniocentesis and chorionic villous sampling: a systematic review. Obstet Gynecol 2007;110:687–94.
Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485–7.
Galbiati S, Smid M, Gambini D, Ferrari A, Restagno G, Viora E, et al. Fetal DNA detection in maternal plasma throughout gestation. Hum Genet 2005;117:243–8.
Chan KC, Zhang J, Hui AB, Wong N, Lau TK, Leung TN, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004;50:88–92.
Davanos N, Spathas DH. Relative quantitation of cell-free fetal DNA in maternal plasma using autosomal DNA markers. Clin Chim Acta 2011;412:1539–43.
Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768–75.
Fleischhacker M, Schmidt B, Weickmann S, Fersching DM, Leszinski GS, Siegele B, et al. Methods for isolation of cell-free plasma DNA strongly affect DNA yield. Clin Chim Acta 2011;412:2085–8.
Clausen FB, Krog GR, Rieneck K, Dziegiel MH. Improvement in fetal DNA extraction from maternal plasma. Evaluation of the NucliSens Magnetic Extraction system and the QIAamp DSP Virus Kit in comparison with the QIAamp DNA Blood Mini Kit. Prenat Diagn 2007;27:6–10.
Fong SL, Zhang JT, Lim CK, Eu KW, Liu Y. Comparison of 7 methods for extracting cell-free DNA from serum samples of colorectal cancer patients. Clin Chem 2009;55:587–9.
Legler TJ, Liu Z, Mavrou A, Finning K, Hromadnikova I, Galbiati S, et al. Workshop report on the extraction of foetal DNA from maternal plasma. Prenat Diagn 2007;27:824–9.
Pinzani P, Salvianti F, Pazzagli M, Orlando C. Circulating nucleic acids in cancer and pregnancy. Methods 2010;50:302–7.
Schmidt B, Weickmann S, Witt C, Fleischhacker M. Improved method for isolating cell-free DNA. Clin Chem 2005;51:1561–3.
Chiu RW, Poon LL, Lau TK, Leung TN, Wong EM, Lo YM. Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 2001;47:1607–13.
Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Analysis of the size distributions of fetal and maternal cell-free DNA by paired-end sequencing. Clin Chem 2010;56: 1279–86.
Macher HC, Noguerol P, Medrano-Campillo P, Garrido-Marquez MR, Rubio-Calvo A, Carmona-Gonzalez M, et al. Standardization non-invasive fetal RHD and SRY determination into clinical routine using a new multiplex RT-PCR assay for fetal cell-free DNA in pregnant women plasma: results in clinical benefits and cost saving. Clin Chim Acta 2012;413:490–4.
Nygren AO, Dean J, Jensen TJ, Kruse S, Kwong W, van den Boom D, et al. Quantification of fetal DNA by use of methylation-based DNA discrimination. Clin Chem 2010;56:1627–35.
Jorgez CJ, Dang DD, Simpson JL, Lewis DE, Bischoff FZ. Quantity versus quality: optimal methods for cell-free DNA isolation from plasma of pregnant women. Genet Med 2006;8:615–9.
Zimmermann B, El-Sheikhah A, Nicolaides K, Holzgreve W, Hahn S. Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma. Clin Chem 2005;51: 1598–604.
Clinical Chemistry and Laboratory Medicine (
CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine.
CCLM welcomes contributions on the progress in fundamental and applied research and cutting-edge clinical laboratory medicine. It is one of the leading journals in the field, with an impact factor of over three.
CCLM is the official journal of nine national clinical societies and associated with EFLM.