When it comes to blood collection and storage, biobankers must ensure that samples yield enough high-quality material to facilitate research goals while maintaining optimal cost performance. Zhou et al. recently evaluated how storage conditions affect DNA quality and quantity.1
To do this, they used biospecimens from 17 donors for three experiments examining the effects of various sample preparation and storage conditions on sample yields and quality:
peripheral blood leukocyte yield and centrifuge speed
buffy coat quality and pretreatment time
DNA quantity/quality and storage time
For the first experiment, the team subjected 3 mL aliquots to centrifugation at five speeds (400 g, 800 g, 1,200 g, 1,600 g and 2,000 g) to evaluate how centrifugal forces affected peripheral blood leukocyte distribution. After removing the plasma, the blood layered into five 300 μL aliquots. The team reported a significant difference in yields from buffy coats centrifued over 800 g (4.2 x 107 cells) versus 400 g (2.8 x 107 cells). Additionally, the quantity of plasma increased with centrifugal force with only 936 μL plasma at 400 g versus 1,214 to 1,280 μL plasma at centrifugal forces over 1,200 g. Finally, ~70% of the peripheral blood leukocytes populated the uppermost two layers of the blood cell pack after centrifugation above 800 g.
After centrifugation at 800 g for 10 minutes, the team stored blood samples at 4ºC before counting peripheral blood leukocytes at four time frames (1 hour, 8 hours, 16 hours and 24 hours). They found no significant difference in buffy coat output (3.8 to 4.7 x 107 cells) or viability between each time point. Additionally, testing for the enzyme LDH, whose release acts as a marker for cell death, revealed no significant difference among the times tested. This indicates that storage at 4ºC for up to 24 hours is unlikely to affect peripheral blood leukocyte viability.
Finally, the team snap froze buffy coat for storage at -80ºC. Using fresh blood as a control (0 hours), they evaluated DNA yield in the frozen samples at three time points (one hour, one week and one month). They found that fresh and one-hour frozen buffy coat yielded similar quantities of DNA (5.3 mg and 4.9 mg, respectively), while freezing for one week or one month produced a decline in yield (3.1 mg and 3.01 mg, respectively). They also confirmed DNA quality and quantity via agarose gel electrophoresis and multiplex PCR and reported no difference for any time point.
Overall, Zhou et al. offer these insights to maximize cost efficiency for biobanking:
Blood sample storage for 24 hours or less at 4ºC does not affect buffy coat yield or cell viability.
DNA extraction should occur using fresh or briefly frozen buffy coat, since the DNA yield can be 1.5 times greater as compared to samples frozen for over one week.
Centrifugation of blood samples at 800 g for 10 minutes and retention of the uppermost 40% of the cell pack preserves at least two-thirds of the peripheral blood leukocytes from whole blood.
As an additional note on the final point, the team cited an example to illustrate how this data could optimize cost performance. They showed that a 3 mL whole blood sample could be pelleted to a 1.8 mL “all-cell-pellet” fraction via centrifugation. Retaining only the 600 to 700 µL topmost portion of the cell pack would preserve over 70% of the cells (representing the majority of the extractable DNA) while saving approximately 60% storage space and maintaining quality control.
1. Zhou, J. et al. (2015) ‘Biostorage and quality control for human peripheral blood leukocytes,’ Biopreservation and Biobanking, 13(1), doi: 10.1089/bio.2014.0094.