Effects of pre-analytical processes on blood samples used in metabolomics studies

Abstract

Every day, analytical and bio-analytical chemists make sustained efforts to improve the sensitivity, specificity, robustness, and reproducibility of their methods. Especially in targeted and non-targeted profiling approaches, including metabolomics analysis, these objectives are not easy to achieve; however, robust and reproducible measurements and low coefficients of variation (CV) are crucial for successful metabolomics approaches. Nevertheless, all efforts from the analysts are in vain if the sample quality is poor, i.e. if preanalytical errors are made by the partner during sample collection. Preanalytical risks and errors are more common than expected, even when standard operating procedures (SOP) are used. This risk is particularly high in clinical studies, and poor sample quality may heavily bias the CV of the final analytical results, leading to disappointing outcomes of the study and consequently, although unjustified, to critical questions about the analytical performance of the approach from the partner who provided the samples. This review focuses on the preanalytical phase of liquid chromatography-mass spectrometry-driven metabolomics analysis of body fluids. Several important preanalytical factors that may seriously affect the profile of the investigated metabolome in body fluids, including factors before sample collection, blood drawing, subsequent handling of the whole blood (transportation), processing of plasma and serum, and inadequate conditions for sample storage, will be discussed. In addition, a detailed description of latent effects on the stability of the blood metabolome and a suggestion for a practical procedure to circumvent risks in the preanalytical phase will be given.

Graphical Abstract

The procedures and potential problems in preanalytical aspects of metabolomics studies using blood samples. Bias in the preanalytical phase may lead to unwanted results in the subsequential studies

Fig. 1

Scheme of the main steps in a clinical metabolomics study. The pre-analytical procedures are given on the left-hand side

Fig. 2

The mass spectrum of polymers shed from Li-heparin Microtainer plastic tubes (Reprint from RCM (2003)17 (1):97–103, license number: 3485190734889). When pure water, blank plasma, and plasma were added to the tubes, typical mass-spectrum patterns could be observed which may cause significant matrix effects on MS

Fig. 3

Evaluation of blood samples placed in iced water after blood drawing. The PCA score plot indicated no significant differences between fresh samples and samples placed at once in iced water for 2 or 4 h. (Reprint from Clinical Chemistry (2013) 59 (5):833–845) (License number: 3485201154760)

Fig. 4

The effect of plasma storage at 37 °C and 4 °C on serotonin, lyso-phospholipids, and choline. LPCs underwent significant changes even at 4 °C. Serotonin and choline were easily affected when placed at 37 °C. Reprinted with permission from Ref. [91] Copyright (2013) American Chemical Society

Fig. 5

The development of a novel sample-preparation method for metabolomics. Using the solvent system MTBE–methanol–water, polar and non-polar metabolites could be effectively simultaneously extracted from a limited amount of tissue. The method can also be used in the preparation of blood samples. (Reprint from J Chromatogr A 1298:9–16, License number 3526350373512)