Effects of blood sample collection and preparation methods on concentrations of glucose and nonesterified fatty acids in dairy cattle

Abstract

Measurement of concentrations of glucose and nonesterified fatty acids (NEFA) in blood is common in nutrition and physiology studies. Proper collection and preparation conditions of the blood have been less well studied in dairy cattle. The objective of this experiment was to determine concentrations of glucose and NEFA in blood prepared with different anticoagulants (heparin vs. EDTA), use of fluoride as a glycolysis inhibitor, time until centrifugation (<30 min to 2.5 h), and plasma versus serum. Blood samples were obtained from 30 lactating cows and 15 milk-fed calves into 5 evacuated test tubes. Three of the tubes contained K3 EDTA and 1 tube contained heparin as anticoagulants to prepare plasma, and the fifth tube was a serum separator tube. One of the EDTA tubes was inverted and divided into 2 tubes containing NaF. One of the tubes with NaF and 1 EDTA tube were centrifuged within 30 min of collection and the others were held on ice for another 2 h before centrifugation. The heparin tube and the serum separator tube were centrifuged within 30 min. Glucose and NEFA were measured in the samples using enzymatic kits. Data were divided into 2 data sets representing normal dairy cow values or elevated values for glucose and NEFA. For glucose concentrations, results indicated that fluoride decreased concentrations, that a 2-h holding time before centrifugation did not affect results, and that serum and EDTA plasma resulted in lower glucose than heparin plasma. For low NEFA concentrations, addition of fluoride to the EDTA tubes resulted in a significant decrease of NEFA concentration. The effect of time sitting before centrifugation was significant for low NEFA samples, but contrary to our expectations, the effect was to decrease NEFA rather than increase it. Heparin as an anticoagulant did not affect NEFA concentrations in the low NEFA samples relative to EDTA. Heparin resulted in lower NEFA than serum in low NEFA samples. Serum and EDTA plasma resulted in similar NEFA concentrations. In the high NEFA samples, experimental power was limited due to the small sample size, but fluoride and time did not affect NEFA concentrations. Heparin tended to result in greater NEFA relative to EDTA. Serum produced greater NEFA values compared with EDTA but did not differ from heparin plasma. While differences among preparation methods were small and of limited biological significance, these results provide guidance for collection and processing of blood samples intended for glucose and NEFA assay.