Real-time PCR evaluation of cell-free DNA subjected to various storage and shipping conditions

Abstract

ABSTRACT. In this study, we attempted to explore the factors affecting the yield of cell-free fetal DNA (cffDNA) obtained from maternal blood samples, including the use of different types of collection tubes, the interval between sample processing, and sample shipping under extreme weather conditions. Blood samples were drawn into K 3 EDTA tubes and cellstabilizing tubes (Streck blood collection tube, BCT) from women pregnant with male fetuses. Real time PCR was used to amplify a β-actin gene fragment to measure the total plasma cell-free DNA concentration, while an SRY gene fragment was used to quantify the cffDNA. The samples in the K 3 EDTA tubes revealed a decreased quantity of SRY after 5 days of transportation, with a median of 25.9 copies/mL (P < 0.01); however, the value remained stable at 33.4 copies/mL in the BCT tubes. We observed a statistically significant increase in stability of the amount of total DNA in the 12798 Q. Wang et al.©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 12797-12804 (2015) blood samples stored in K 3 EDTA tubes (P < 0.01) and transportated under extreme outdoor temperatures (-20°-0°C) than that of the control values. These results indicate that it could be possible to avoid the presence of excess maternal DNA in samples shipped under extreme weather conditions for no more than 2 days, by collecting the blood samples in BCT tubes.

Fetal fraction evaluation in non-invasive prenatal screening (NIPS)

“…Within this time interval we saw no correlation between shipping time and FF (Supplemental Fig. 3), confirming previous reports [2] and fitting within manufacturer guidelines (up to 14 days at 6-37°C) when using Streck tubes.…”

“…Indeed, some laboratories may not be checking FF or using optimal methods for detection that could potentially provide false-negative results to patients [1]. Technical factors that can influence FF and its estimation are sample handling [2] and the choice of bioinformatics tools (reviewed in [3]) [4]. Multiple biological factors have been identified that affect FF, including gestational age [4][5][6][7][8][9], weight and/or body mass index (BMI) [6][7][8][9][10][11][12], trisomies [7,[10][11][12][13], fetal crown-rump length [6,10,12], serum pregnancy-associated plasma protein-A [6,10,12], serum free β-human chorionic gonadotropin [6,10,12], hypertension [7], twins [7], smoking [10], and assisted conception [8,12].…”

Effect of sample type on plasma concentrations of cell‐free DNA and nucleosomes in dogs

“…Plasma samples are preferable to serum because release of DNA from leukocytes following collection can falsely increase measured concentrations 29–31. Other factors that may affect cfDNA concentrations are time between collection and processing,4 32 centrifugation methods,29 and storage conditions 29 33…”

Current status and future perspectives of circulating cell-free DNA methylation in clinical diagnostics

“…Streck, Inc. (Omaha, NE, USA) developed blood collection tubes (Cell-Free DNA BCT ® ) which contain a formalin-releasing reagent leading to a slow fixation of the DNA and the cells in a blood sample. These collection tubes allow for storage of blood samples for several days at ambient temperature before starting the plasma preparation procedure [35][36][37][38][39]. While this would dramatically facilitate the sample logistic needed for a centralized high-throughput testing of methylated ccfDNA, these tubes are still expensive and a cost-driving factor in the overall complex procedure.…”