Abstract Cell-free DNA (cfDNA) results from cell death and/or cellular secretion. In human medicine, it has been commonly used as a biomarker and liquid biopsy to study physiological and pathological events, such as pregnancy and cancer. During pregnancy, cfDNA from the mother and the fetus circulates in maternal blood, and 10 mL of blood is sufficient to characterize these markers. Although cfDNA has been studied as a biomarker in domestic animals, research on stability and storage requirements in these species is scarce. As a result, researchers often rely on human protocols or non-standardized methods for their experiments. Our study aims to optimize the pre-analytical blood sample processing protocols for cfDNA analysis in beef cattle. Angus pregnant cows (n = 28) were sampled for whole blood at day 180 of gestation. Blood samples were taken using two types of tubes: EDTA and PAXgene Blood ccfDNA Tubes. The second tubes are designed for collecting, transporting, and storing human whole blood samples, as well as stabilizing cfDNA. Each tube type was stored for 0, 6, 12, or 24 hours at either 0 °C or 20 °C. Samples were then centrifuged to separate the plasma. After cfDNA extraction, total DNA and cfDNA concentration were measured using a cfDNA Screen Tape Assay. Data was analyzed using Proc MIXED (SAS), including the type of tube, storage time, temperature, and their interactions as fixed effects. The tube type, temperature, and storage time influenced total DNA. Additionally, all two-way and three-way interactions were significant (p 0.001) for total DNA concentration. Due to the significant three-way interaction, the results are presented based on their combined effects. The total DNA levels in PAXgene tubes increased (p 0.001) at 12h and 24h when stored at 0 °C. In contrast, EDTA tubes, total DNA levels increased (p 0.001) at 20 °C storage, at 6h, 12h, and 24h, demonstrating better total DNA stability when blood was stored in EDTA tubes at 0 °C and PAXgene tubes at 20 °C. Comparably, for cfDNA, all interactions, including the three-way interaction, were also significant (p 0.001). The concentration of cfDNA remained stable in both tubes at 0h and 6h. At 12h, samples stored at 20 °C in both tubes increased their concentration (p 0.001), and at 24h, both temperatures in the PAXgene tubes presented a three-time-fold increase. However, at 24h, EDTA tubes at 0 °C remained stable, and EDTA at 20 °C presented a three-time-fold increase (p 0.001) similar to PAXgene tubes. We conclude that blood samples stored in EDTA tubes at 0 °C for later cfDNA analysis in beef cattle performed better than PAXgene in total DNA and cfDNA stability. Moreover, PAXgene tubes do not offer equal stability for cfDNA in cattle as in human samples.
Duarte et al. (Wed,) studied this question.