Comparison of glucose concentrations in samples frozen for varying durations and subjected to freeze-thaw cycles using 2 assay systems

Although laboratory best practices recommend that total storage time and number of freeze-thaw cycles should be minimized before analysis of metabolites within plasma, the specific impacts of storage time and freeze-thaw cycles on apparent glucose concentrations in bovine plasma have not been well quantified. Additionally, within the field of dairy science, multiple assay systems for glucose concentration analysis are used, yet these assays have not been directly compared with each other. Thus, the first objective was to investigate the stability of glucose in samples that have been frozen for an increased duration or frozen and thawed up to 4 times. The second objective was to investigate the agreement between 2 assay systems for glucose measurement: the peroxidase and glucose oxidase (PGO) assay system and the hexokinase reaction (HK) assay system. Blood samples were collected into sodium fluoride (NaF) tubes from 166 dairy cows, across different lactation stages, on 3 farms over a 2-d interval. After centrifugation, plasma was separated into 4 aliquots. One aliquot was used to determine the impacts of up to 4 freeze-thaw cycles, and the first measurement from this aliquot along with the other 3 aliquots were used to investigate potential glucose degradation in aliquots frozen for different lengths of time, including 2, 3, 4, and 16 wk. All samples were analyzed using both the PGO and HK assay systems. Bland–Altman plots, Pearson correlations, and paired t-tests were used to compare the effects of storage time and freeze-thaw cycles. Additionally, a linear mixed model with the fixed effect of either storage time or freeze-thaw cycle and the random effect of cow within farm was used to consider the effect of each of these factors on measured glucose concentrations. For comparisons between assay systems, a linear mixed model was used with the fixed effect of assay system, time, frozen or freeze-thaw cycles depending on the model, their interaction, and cow within farm as a random effect. Storage time and freeze-thaw cycles influenced glucose concentration measured by the PGO and HK. Concentrations of glucose both increased and decreased with increasing storage time and freeze-thaw cycles and compared with the initial quantification. Apparent glucose concentration was more variable across all aliquots when the HK assay (± 0.14 mmol/L) was used compared with the PGO assay (± 0.06 mmol/L). The HK assay continuously yielded lower concentrations of glucose compared with the PGO assay. Overall, glucose concentrations can be variable over different storage times and freeze-thaw cycles; however, they cannot be compared across the PGO and HK assay systems.