Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma

Background: Recently, apoptotic cells have been found in plasma obtained by centrifugation of blood from pregnant women, raising the question of what constitutes plasma and whether plasma is truly cell free. We compared the e ffects of different blood-processing protocols on the quantification, DNA c omposition, and day-to-day fluctuation of fetal and total DNA in maternal p lasma. Methods: Blood samples were collected from healthy pregnant women. The bloo d sample from each individual was simultaneously processed by different mea ns, including the following: Percoll separation, centrifugation, microcentr ifugation, and filtration. The resulting plasma aliquots were subjected to real-time quantitative amplification of the beta -globin (for total DNA) an d SRY (for fetal DNA) genes. The differences in the beta -globin and SRY DN A concentrations and the degree of variation between the various plasma ali quots were assessed statistically. Results: Different protocols of blood processing significantly affected the quantification and the day-to-day fluctuation of total (P < 0.001), but no t fetal (quantification, P = 0.336; fluctuation, P = 0.206), DNA in materna l plasma. The quantitative difference could be attributed to the fact that efficacies of different protocols for generating cell-free plasma vary. Pro cessing blood samples by centrifugation followed by filtration or microcent rifugation is effective in producing cell-free plasma. Conclusions: Standardization in plasma-processing protocols is needed for m aternal plasma DNA analysis, especially for quantification of total DNA in maternal plasma. Such preanalytic factors may also affect other application s of plasma DNA analysis. (C) 2001 American Association for Clinical Chemis try.