Effect of storage time, temperature, antioxidant and thawing on fatty acid composition of plasma, serum and red blood cells - A pilot biobank study.
Author(s): Araujo P, Bjørkkjær T, Frøyland L, Waagbø R
Publication: Clin Biochem, 2018, Vol. 52, Page 94-105
PubMed ID: 29054439 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of the addition of butylated hydroxytoluene (BHT) or methanol on the stability of fatty acids in plasma, serum, and red blood cells during frozen storage at -20˚C and -80˚C and in red blood cells during freeze-thaw cycling.
Conclusion of Paper
Levels of stearic acid and eicosapentaenoic acid in fresh serum were affected by the addition of BHT or methanol, but there was no effect of BHT or methanol addition on levels of fatty acids in plasma or red blood cells. Regardless of storage temperature, fatty acid levels were stable in plasma with or without BHT and were stable in serum with BHT for up to 52 weeks, in red blood cells with BHT for 26 weeks, and in serum or red blood cells without BHT for 13 weeks. Fatty acid levels in red blood cells without BHT were stable through four freeze-thaw cycles at -20˚C, but only one freeze-thaw cycle at -80˚C. However, fatty acids levels were stable through 5 cycles at -20˚C or 6 cycles at -80˚C when BHT was added to red blood cells.
Studies
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Study Purpose
This study investigated the effects of the addition of BHT on the stability of fatty acids in plasma, serum, and red blood cells during frozen storage at -20˚C or -80˚C and in red blood cells during freeze-thaw cycling. Blood was collected from a single healthy woman into serum tubes (Vacutainer SST) and K2EDTA tubes. Serum tubes were allowed to clot for 30 min, centrifuged at 1500 x g for 15 min, and then the supernatant was transferred to new tubes. K2EDTA tubes were centrifuged at 1500 x g for 15 min to separate the plasma, leukocytes, and red blood cells. The plasma was transferred to a new tube and recentrifuged at 1500 x g before being placed in a pooling tube. The red blood cells were not purified further after removal of the leukocyte layer. Two replicate plasma and serum specimens were stored at -20˚C and -80˚C for 13, 26, and 52 weeks in the presence or absence of BHT in methanol or methanol. RBCs were stored for 4, 6, 9, 13, 26, and 52 weeks at -20˚C and -80˚C in the presence or absence of BHT in methanol or methanol alone. The effect of thawing of RBC was examined by thawing specimens after 2, 4, 6, 9, 11, 13 and 26 weeks. At each time-point, the RBCs were thawed and analyzed and the remainder of the specimen was returned to storage. FAME analysis was conducted using a Autosystem XL Gas Chromatograph.
Summary of Findings:
Fresh serum had lower levels of stearic acid and eicosapentaenoic acid when BHT or methanol were added than in their absence, but there was no effect of BHT or methanol addition on levels of fatty acids in plasma or red blood cells. Fatty acid levels were unaffected by frozen storage of plasma for up to 52 weeks at either temperature, regardless of the addition of BHT and remained within 15% of those in fresh plasma. Fatty acid levels were stable in serum with BHT for 52 weeks at either temperature, but in the absence of BHT, significant declines in 4 fatty acids were noted after 26 weeks at -20˚C (-17% to -32%, P<0.05) and a significant decline in levels of 8 fatty acids occurred after 26 weeks at -80˚C (-19% to -50%, P<0.05). Fatty acids were stable in red blood cells stored with BHT for up to 26 weeks at either temperature, but without BHT, levels of 5 fatty acids increased (33-84%, P<0.05) and 6 decreased (-54 to -62%, P<0.05) when stored at -20 ˚C for 26 weeks and levels of 5 fatty acids increased (16-100%, P<0.05) and 2 decreased (-46% to -60%, P<0.05) when stored at -80˚C for 26 weeks. Fatty acid levels in red blood cells without BHT were stable for four freeze-thaw cycles at -20˚C, but levels of two fatty acids were increased (50-60%, P<0.05) and levels of one fatty acid were decreased (-54%, P<0.05) after the fifth freeze-thaw cycle. Freeze-thaw cycling at -80˚C of red blood cells without BHT resulted in a 19% decline in one fatty acid after two cycles and increases in 5 fatty acids (32-123%, P<0.05) after three cycles. In contrast, fatty acids levels were stable through five cycles at -20˚C or six cycles at -80˚C when RBCs were stored with BHT.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Small molecule GC Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Red blood cells
Serum
Storage Storage temperature -20˚C
-80˚C
Storage Storage duration 0 weeks
4 weeks
6 weeks
9 weeks
13 weeks
26 weeks
52 weeks
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
3 cycles
4 cycles
5 cycles
6 cycles
7 cycles
Biospecimen Preservation Fixative additive/buffer Butylated hydroxytoluene
Methanol
None