Impact of Analytical Bias in Metabonomic Studies of Human Blood Serum and Plasma

Abstract

Concurrent with the explosion in the number of publications reporting biomarker discovery by profiling technologies, such as proteomics and pattern recognition, has been the increase in evidence highlighting the susceptibility of these approaches to analytical and experimental bias. The work presented here addresses these timely issues by delivering a detailed characterization of the effect of common sources of bias in clinical studies on serum and plasma profiles generated by a key technology in metabonomics, NMR spectroscopy. Specifically, differences in composition when blood samples were collected onto and in the absence of ice, over a series of serum-clot contact times, the stability of NMR-prepared samples over time and the effect on the metabolic profile of freeze-thawing were examined. While differences between individuals were far greater than variation from any other experimental factor, each of the conditions examined did cause slight alterations to the NMR profile that could produce a systematic bias. Variation due to clotting time caused changes in energy metabolites, which were delayed by ice with no other spectral effects. Room-temperature stability and hence NMR spectral repeatability were high (<1% intrasample variation). Higher molecular weight species such as lipoproteins were more susceptible to the variations present in the examined factors. These observations have implications for profiling study design, and hence, our results form a new and valuable resource for those attempting clinical metabolic profiling, for regulatory agencies involved in the licensing of clinical tests and in the generation of international reporting standards for metabonomics.

Optimized Sample Handling Strategy for Metabolic Profiling of Human Feces

“…The metabolites assigned from the 1 H NMR spectra are summarized in Table S2. High interpersonal variations have been previously reported not only in the metabolic profiles of urine 16 and plasma 17 , but also in the gut microbial composition 18 and fecal metabolites 11 , implicating that the genetic background and environmental diversity significantly contribute to the metabolic phenotype of the host. Aside from being requested to abstain from alcohol for 24 h prior to fecal sample donation, the donors enrolled in the study were not subjected to any dietary restrictions, which could enhance the interpersonal variations observed.…”

The Impact of Delayed Storage on the Measured Proteome and Metabolome of Human Cerebrospinal Fluid

“…To detect reliable molecular biomarkers, it is imperative to handle biological fluids according to standardized procedures and to evaluate the effects of preanalytical parameters on the final result to avoid artifacts (1,2 ). Earlier studies on urine, plasma, and cerebrospinal fluid (CSF) 9 have shown that sample handling can affect the stability of proteins as well as metabolites (3)(4)(5)(6)(7)(8)(9)(10)(11). Standardized sample handling is also important when trying to compare results between different laboratories (12)(13)(14).…”

Standard reporting requirements for biological samples in metabolomics experiments: mammalian/in vivo experiments

“…For functional genomic studies to specifically address the influence of the strain background two papers were chosen that demonstrate the profound influence the genetic background has on the metabolic function and phenotype of a genetically modified organism (Hough et al 2002;Sam et al 2001). Finally a combination of clinical trials and studies of human metabolism where samples have been taken under less strict control of external variables were also considered (Brindle et al 2002;Poston et al 2006;Wolff et al 2004;Siu et al 2006;Townsley et al 2006;Khor et al 2006;Teahan et al 2006). These resultant reporting requirements were then compared with the information captured by other standards initiatives including MIAME (Minimum Information About a Microarray Experiment; Brazma et al 2001;Quackenbush 2004), (minimum information about a genome sequence (MIGS); http://www.genomics.ceh.ac.uk/genomecatalogue; Field et al 2006), CEBS and the human proteome organisation-proteomics standard initiative's (HuPO-PSI; minimum information about a proteomics experiment (MIAPE) ;Taylor 2006;Orchard et al 2003;Orchard et al 2005).…”