Effects of sample handling, processing, storage, and hemolysis on measurements of key energy metabolites in ovine blood

Abstract

Four experiments were conducted using mature Suffolk ewes to evaluate the effects of blood sample handling, processing and storage on measurements of the energy metabolites, β-hydroxybutyrate, total cholesterol, glucose, l-lactate, nonesterified fatty acid (NEFA), and triacylglycerol. In experiment 1 the effects of anticoagulants on metabolite measurements and packed-cell volume (PCV) were evaluated. Blood samples ( n=12) were collected into one of four evacuated blood-collecting tubes: no anticoagulant (SER; yields serum), and plasma-yielding sodium heparin (HEP), sodium fluoride and potassium oxalate (NAF), and tripotassium ethylenediaminetetraacetic acid (K 3EDTA). Anticoagulant affected ( P<0.05) metabolite values, with total cholesterol, triacylglycerol, and l-lactate highest in SER, and glucose highest in HEP; NEFA and β-hydroxybutyrate measurements were not affected ( P>0.10) by anticoagulant. In addition, among the plasma-yielding tubes, PCV was highest in HEP and lowest in NAF ( P<0.01). Experiment 2 investigated the effects of repetitive freezing-thawing cycles of plasma on metabolite levels. Blood samples ( n=20) were collected using NAF tubes, and plasma was stored at −20 °C and thawed in a water bath (21 °C) 0, 1, 2, and 3 times within 18 h after collection. Compared with fresh samples (Thaw 0), by thaw 2, l-lactate increased ( P<0.01) 5%, and glucose and total cholesterol decreased ( P<0.001) 13 and 4%, respectively. Plasma NEFA increased 10% ( P<0.01) between thaws 0 and 1, but returned to fresh levels (Thaw 0) with subsequent freeze-thaw cycles ( P<0.05). Plasma β-hydroxybutyrate decreased ( P<0.01) between thaws 0 and 1, but there was no further decline with subsequent freeze-thaw cycles ( P<0.05). Experiment 3 evaluated the effects of plasma storage temperature (−20 °C versus −80 °C) and length (0–180 days) on metabolite levels in blood samples ( n=12) collected in NAF tubes. All plasma metabolites were affected by storage length (day effect, P<0.01) but only total cholesterol values were affected by storage temperature, with values being higher in plasma stored at −20 than −80 °C ( P<0.01). Glucose measurements were higher in samples stored at −20 °C for up to 30 days, but were higher thereafter in samples stored at −80 °C (storage length × temperature effect, P<0.01). Experiment 4 evaluated the effects of anticoagulant (SER versus NAF) and whole blood storage (4 °C) for 0, 1, 3, and 7 days on metabolite concentrations. Glucose was higher ( P<0.0001) in NAF, possibly the result of the presence of the glycolytic inhibitor, sodium fluoride, whereas l-lactate, NEFA, total cholesterol and β-hydroxybutyrate were higher in SER ( P<0.01). Total cholesterol, l-lactate, and NEFA increased, whereas β-hydroxybutyrate decreased with days in storage ( P<0.01). Development of hemolysis in the samples artifactually elevated glucose and NEFA values by as much as 41 and 230%, respectively. Attention to proper blood handling, processing, and storage procedures, and avoidance of hemolysis are important in blood clinical analyses and in the proper interpretation of experimental results.