BACKGROUND: Peripheral blood leukocytes (PBLs) are the main source of DNA in blood samples. A common protocol is to store buffy coat specimens for future DNA isolation, and such samples may remain in frozen storage for years. However, the currently available methods to maintain buffy coat specimens can be optimized for better quality and cost efficiency. STUDY DESIGN AND METHODS: Seventeen donors (aged 24-34 years) were enrolled in this study. Initially, five centrifugal speeds were chosen to examine the distribution of PBLs in the cell layer; the buffy coat was then harvested to evaluate the cell quantity and viability after storing at 4°C for various times. Finally, the buffy coat was isolated, snap frozen, and kept at -80°C for 1 hour, 1 week, or 4 weeks until the DNA was extracted. Agarose gel electrophoresis and multiplex PCR were used to verify DNA integrity and amplifiability. RESULTS: There was a linear positive correlation between the amount of fresh PBLs and the DNA yield. At least 70% of PBLs were collected in the uppermost 40% of the cellular material when the centrifugal force was over 800 g compared to 400 g. Storing blood at 4°C for no more than 24 hours did not have an effect on the amount of PBLs or their viability. In addition, the amount of extracted DNA was decreased in the frozen buffy coat that was stored for more than 7 days, though the DNA quality was acceptable. CONCLUSION: DNA should be extracted from fresh buffy coat samples as soon as possible after collection, especially for very important samples. Retaining the upper 40% of the cell pack of blood instead of whole blood could improve the storage efficiency of biobanking such samples. Our study provides a recommended option for blood collection and processing protocols in biobanking.
BACKGROUND: Peripheral blood leukocytes (PBLs) are the main source of DNA in blood samples. A common protocol is to store buffy coat specimens for future DNA isolation, and such samples may remain in frozen storage for years. However, the currently available methods to maintain buffy coat specimens can be optimized for better quality and cost efficiency. STUDY DESIGN AND METHODS: Seventeen donors (aged 24-34 years) were enrolled in this study. Initially, five centrifugal speeds were chosen to examine the distribution of PBLs in the cell layer; the buffy coat was then harvested to evaluate the cell quantity and viability after storing at 4°C for various times. Finally, the buffy coat was isolated, snap frozen, and kept at -80°C for 1 hour, 1 week, or 4 weeks until the DNA was extracted. Agarose gel electrophoresis and multiplex PCR were used to verify DNA integrity and amplifiability. RESULTS: There was a linear positive correlation between the amount of fresh PBLs and the DNA yield. At least 70% of PBLs were collected in the uppermost 40% of the cellular material when the centrifugal force was over 800 g compared to 400 g. Storing blood at 4°C for no more than 24 hours did not have an effect on the amount of PBLs or their viability. In addition, the amount of extracted DNA was decreased in the frozen buffy coat that was stored for more than 7 days, though the DNA quality was acceptable. CONCLUSION: DNA should be extracted from fresh buffy coat samples as soon as possible after collection, especially for very important samples. Retaining the upper 40% of the cell pack of blood instead of whole blood could improve the storage efficiency of biobanking such samples. Our study provides a recommended option for blood collection and processing protocols in biobanking.