Effects of processing and storage on the measurement of nitrogenous compounds in ovine blood

Abstract

Four experiments were conducted to evaluate effects of processing and storage of sheep blood samples on measurements of ammonia N (AMN), urea N (URN), total protein (TP), albumin (ALB), total globulin (GLB), and the non-albumin non-globulin protein fraction (NAG). Experiment 1 compared the effects of using different anticoagulants. Blood samples ( n = 15) were collected via jugular venipuncture into one of four evacuated tubes: no anticoagulant, serum; sodium heparin, plasma; sodium fluoride and potassium oxalate, plasma; tripotassium-ethylenediaminetetraacetic acid (EDTA), plasma. Anticoagulant affected ( P < 0.0001) metabolite values, with TP, ALB, and GLB being higher in heparinized plasma, and URN, NAG, and AMN highest in serum. In addition, blood packed-cell volume was 12% and 15% higher ( P < 0.0001) in samples collected in heparin and EDTA compared with samples collected in sodium fluoride and potassium oxalate. Experiment 2 investigated the effects of repetitive freezing and thawing (0–3 thaw cycles) of plasma samples ( n = 15 blood samples collected in tubes containing sodium fluoride and potassium oxalate). Repetitive freezing and thawing decreased ( P < 0.0001) URN and NAG by 21% and 54%, respectively, the latter as a result of a 4–13% increase ( P < 0.0006) in ALB and GLB values. Experiment 3 evaluated the effects of storage length (0–26 weeks) and freezing temperatures (−20 °C vs. − 80 °C) on plasma samples ( n = 16–20 blood samples collected in tubes containing sodium fluoride and potassium oxalate). All nitrogenous metabolites were affected by length of storage ( P < 0.0001). Plasma AMN, URN, TP, and NAG were affected (0.0001 < P < 0.10) by storage temperature, and AMN, TP, GLB, and NAG measurements exhibited a temperature × day interaction ( P < 0.05). In general, TP and NAG values declined with storage length, whereas URN, GLB, and AMN values increased. Plasma AMN increased 74% by Week 1 and 160% by Week 9, and samples stored at −20 °C were 14% higher than samples stored at −80 °C ( P < 0.0001). Experiment 4 assessed the effects of storage of whole blood samples ( n = 15 blood samples collected for serum) at 4 °C for 0–7 days. Serum URN and GLB decreased ( P < 0.0001), and TP and AMN increased (0.0001 < P < 0.02) with increasing refrigeration time. Standardized analytical procedures must be followed to obtain reliable nitrogenous metabolite measurements.