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Evaluation of sample stability and automated DNA extraction for fetal sex determination using cell-free fetal DNA in maternal plasma.

Ordoñez E, Rueda L, Cañadas MP, Fuster C, Cirigliano V - Biomed Res Int (2013)

Bottom Line: An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure.Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses.This would allow shipping to a central reference laboratory almost from anywhere in Europe.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica Molecular, Labco Diagnostics, c/Londres 28, 08029 Barcelona, Spain ; Unitat de Biologia, Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.

ABSTRACT

Objective: The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma.

Methods: A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR.

Results: Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses.

Conclusion: We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

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Genomic equivalents of cfDNA in 50 samples from male fetuses extracted with COBAS Ampliprep and QIAGEN Viral DSP procedures.
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fig1: Genomic equivalents of cfDNA in 50 samples from male fetuses extracted with COBAS Ampliprep and QIAGEN Viral DSP procedures.

Mentions: As shown in Table 1, manual DNA extraction resulted in lower Ct values for the SRY amplification (mean Ct = 36,59) than automated procedure (mean Ct = 37,74). Overall, cfDNA amounts were higher using manual extraction compared with the automated system, with a mean quantity of cfDNA of 259.43 GE/mL of plasma (range: 61,05–725.33) and 107,35 GE/mL (range: 10,28–327,06), respectively (Figure 1).


Evaluation of sample stability and automated DNA extraction for fetal sex determination using cell-free fetal DNA in maternal plasma.

Ordoñez E, Rueda L, Cañadas MP, Fuster C, Cirigliano V - Biomed Res Int (2013)

Genomic equivalents of cfDNA in 50 samples from male fetuses extracted with COBAS Ampliprep and QIAGEN Viral DSP procedures.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814069&req=5

fig1: Genomic equivalents of cfDNA in 50 samples from male fetuses extracted with COBAS Ampliprep and QIAGEN Viral DSP procedures.
Mentions: As shown in Table 1, manual DNA extraction resulted in lower Ct values for the SRY amplification (mean Ct = 36,59) than automated procedure (mean Ct = 37,74). Overall, cfDNA amounts were higher using manual extraction compared with the automated system, with a mean quantity of cfDNA of 259.43 GE/mL of plasma (range: 61,05–725.33) and 107,35 GE/mL (range: 10,28–327,06), respectively (Figure 1).

Bottom Line: An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure.Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses.This would allow shipping to a central reference laboratory almost from anywhere in Europe.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica Molecular, Labco Diagnostics, c/Londres 28, 08029 Barcelona, Spain ; Unitat de Biologia, Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.

ABSTRACT

Objective: The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma.

Methods: A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR.

Results: Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses.

Conclusion: We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

Show MeSH