Does centrifugation matter? Centrifugal force and spinning time alter the plasma metabolome

Abstract

BackgroundCentrifugation is an indispensable procedure for plasma sample preparation, but applied conditions can vary between labs.AimDetermine whether routinely used plasma centrifugation protocols (1500×g 10 min; 3000×g 5 min) influence non-targeted metabolomic analyses.MethodsNuclear magnetic resonance spectroscopy (NMR) and High Resolution Mass Spectrometry (HRMS) data were evaluated with sparse partial least squares discriminant analyses and compared with cell count measurements.ResultsBesides significant differences in platelet count, we identified substantial alterations in NMR and HRMS data related to the different centrifugation protocols.ConclusionAlready minor differences in plasma centrifugation can significantly influence metabolomic patterns and potentially bias metabolomics studies.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-016-1109-3) contains supplementary material, which is available to authorized users.

A Systematic Evaluation of Blood Serum and Plasma Pre-Analytics for Metabolomics Cohort Studies

“…A significant impact on the serum and plasma concentrations of lactate and glucose is only observed when increasing the processing delay to 6 h at room temperature, while serum and plasma profiles of acceptable quality are still obtained for samples kept at 22 °C for 1 h, or at 4 °C for 6 h. Our systematic investigation also shows that a short delay (<1 h) at room temperature between preparation (after centrifugation) and −80 °C freezing has no significant influence on the multivariate metabolomic profiles of serum and plasma, while previous studies have reported a gradual degradation over longer time periods (several hours) for blood samples, notably regarding lipids profiles, in the presence of oxygen at room temperature [26,28,32]. Peculiarly, our results concerning centrifugal parameters do not seem in clear agreement with recent observations from Lesche et al, where changes in the centrifugation protocol (combined variations of centrifugal force and spinning time) significantly influenced plasma metabolomics patterns at room temperature [34]. Under our tested variant protocols, serum and plasma metabolic profiles were shown here invariant to both of these parameters, as well as the centrifugation temperature, when studied individually.…”

Metabolic profiling of listeria rhombencephalitis in small ruminants by 1H high‐resolution magic angle spinning NMR spectroscopy

“…The effect on signal loss of the small metabolite peaks was minor; however, the long interpulse spacing of 2 ms was associated with residual J ‐evolution hampering, in our case, the interpretation of the methine resonance of Lac. In the meantime, we are using the “project” sequence to avoid these artifacts …”

“…In the meantime, we are using the "project" sequence to avoid these artifacts. 39,[59][60][61] Finally, the NMR experiment was performed at a nominal temperature of 285 K and the acquisition time was 82 min, which may have led to metabolic changes during the experiment due to ongoing enzymatic activity, and may be reflected by the presence of Ace in the spectra of both the diseased and control groups. However, as the conditions were identical for all biopsy samples, the statistical analysis concerning group differences was probably unaffected by this, and the differences are very unlikely to be due to degradation processes, but rather probably reflect differences that are also present in vivo.…”

Nutrimetabolomics: An Integrative Action for Metabolomic Analyses in Human Nutritional Studies

“…Although manufacturers recommend centrifugation time and speed, still different centrifugation protocols alter the biochemistry of the samples through different amount of platelets remaining in the plasma. Indeed, significant differences in platelet count, together with alterations in NMR and HRMS metabolomes, related to the different centrifugation protocols were recently found . Yazigi Junior et al .…”

“…As a protocol at 3000 × g for 5 min resulted in lower platelet counts, the authors assumed that the centrifugal force recompenses the effect of the centrifugation time. Moreover, in the NMR analyses, the plasma content of free glutamine, as well as of different lipid classes (i.e., glycerophosphocholines and sphingomyelins) in untargeted UPLC–QTOF‐MS analyses, was associated with the centrifugation conditions …”