Variability of alpha-tocolpherol values associated with procurement, storage, and freezing of equine serum and plasma samples

Recent evidence concerning the pathogenesis of equine degenerative myeloencephalopathy indicated that low blood alpha-tocopherol values are a factor in the disease process. Variables that could be introduced by a veterinarian procuring, transporting, or storing samples were evaluated for effects on alpha-tocopherol concentration in equine blood. These variables included temperature; light; exposure to the rubber stopper of the evacuated blood collection tube; hemolysis; duration of freezing time, with and without nitrogen blanketing; and repeated freeze/thaw cycles. It was found that hemolysis caused the greatest change in high-performance liquid chromatography-measured serum alpha-tocopherol values, with mean decrease of 33% (P < 0.001). Lesser, but significant (P < 0.01) changes in serum alpha-tocopherol values were an approximate 10% decrease when refrigerated blood was left in contact with the red rubber stopper of the blood collection tube for 72 hours and an approximate 5% increase when blood was stored at 20 to 25 C (room temperature) for 72 hours. Repeated freeze/thaw cycles resulted in a significant (P < 0.05) 3% decrease in alpha-tocopherol values in heparinized plasma by the third thawing cycle. Freezer storage for a 3-month period without nitrogen blanketing resulted in slight (2%) decrease in mean serum alpha-tocopherol values, whereas values in serum stored for an identical period under nitrogen blanketing did not change. A significant (P < 0.001) mean decrease (10.3%) in alpha-tocopherol values was associated with freezer (-16 C) storage of nitrogen blanketed serum for 6 months. Comparison of alpha-tocopherol values in serum vs heparinized plasma vs EDTA-treated plasma resulted in serum values being significantly (P < 0.001) higher (approx 4%) than values in either type of plasma. It was concluded that the optimal method for storing equine blood samples prior to alpha-tocopherol analysis is in an upright position in a refrigerator for up to 72 hours. If a longer period is needed prior to analysis, it is recommended that the serum or plasma be separated from blood, blanketed with nitrogen gas, and frozen in the smallest possible vial. The alpha-tocopherol in these samples should be stable at -16 C for at least 3 months.