Dataset Information

Ethanol contamination of cerebrospinal fluid during standardized sampling and its effect on (1)H-NMR metabolomics.

ABSTRACT: Standardization of body fluid sampling, processing and storage procedures is pivotal to ensure data quality in metabolomics studies. Yet, despite strict adherence to standard sampling guidelines, we detected variable levels of ethanol in the (1)H-NMR spectra of human cerebrospinal fluid (CSF) samples (range 9.2?×?10(-3)-10.0 mM). The presence of ethanol in all samples and the wide range of concentrations clearly indicated contamination of the samples of some sort, which affected the (1)H-NMR spectra quality and the interpretation. To determine where in the sampling protocol the ethanol contamination occurs, we performed a CSF sampling protocol simulation with 0.9 % NaCl (saline) instead of CSF and detected ethanol in all simulation samples. Ethanol diffusion through air during sampling and preparation stages appeared the only logical explanation. With a bench study, we showed that ethanol easily diffuses into ex vivo CSF samples via air transmission. Ethanol originated from routinely used skin disinfectants containing ethanol and from laboratory procedures. Ethanol affected the CSF sample matrix at concentrations above ~9.4 mM and obscured a significant part of the (1)H-NMR spectrum. CSF sample preparation for (1)H-NMR-based metabolomics analyses should therefore be carried out in a well-ventilated atmosphere with laminar flow, and use of ethanol should be avoided.

SUBMITTER: van der Sar SA

PROVIDER: S-EPMC4446525 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Ethanol contamination of cerebrospinal fluid during standardized sampling and its effect on (1)H-NMR metabolomics.

van der Sar Sonia A SA Zielman Ronald R Terwindt Gisela M GM van den Maagdenberg Arn M J M AM Deelder André M AM Mayboroda Oleg A OA Meissner Axel A Ferrari Michel D MD

Analytical and bioanalytical chemistry 20150503 16

Standardization of body fluid sampling, processing and storage procedures is pivotal to ensure data quality in metabolomics studies. Yet, despite strict adherence to standard sampling guidelines, we detected variable levels of ethanol in the (1)H-NMR spectra of human cerebrospinal fluid (CSF) samples (range 9.2 × 10(-3)-10.0 mM). The presence of ethanol in all samples and the wide range of concentrations clearly indicated contamination of the samples of some sort, which affected the (1)H-NMR spe ...[more]

PMID: 25935669

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Project description:Background: Blood contamination due to traumatic lumbar puncture presents a diagnostic pitfall in cerebrospinal fluid (CSF) analysis. It is controversially discussed if phagocytosis of erythrocytes which can be found in the CSF after subarachnoid hemorrhage can also develop in vitro in the presence of artificial blood contamination. Furthermore, there is no consensus about the acceptable amount of artificial blood contamination on CSF protein results. Methods: Two measurement series were performed in order to investigate the role of artificial blood contamination on the possible development of erythrophages and siderophages in the CSF: (1) blood contamination was simulated in vitro by adding blood into the CSF. (2) CSF was investigated when blood contamination occurred during a traumatic lumbar puncture. In both types of experiments, CSF including blood was incubated for 24 h and for 72 h at room temperature or at 4°C. In the third measurement series, the effects of artificial blood contamination on CSF protein results were investigated. Blood contamination was simulated in vitro by adding different amounts of blood ending up with five different samples containing erythrocyte counts of 2,500, 5,000, 7,500, 10,000, and 20,000 per μl CSF. Results: Cytological examination revealed no evidence of erythrophages or siderophages in vitro. In contrast, already a low blood contamination (2,500 erythrocytes/μl CSF) led to false pathological results of total protein and albumin. Along with increasing amounts of blood, the frequency of false pathological protein results increased. A blood contamination of 5,000 erythrocytes/μl CSF resulted in a false positive intrathecal IgM production in nearly every fifth patient. In contrast, blood contamination with 5,000 erythrocytes/μl CSF was the acceptable amount of blood which did not lead to a false positive intrathecal synthesis of IgG and IgA. Conclusion: Erythrophages and siderophages do not develop in vitro. An extensive diagnostic work up for the source of blood in the CSF should be performed when erythrophages or siderophages are found in the CSF. The contamination of CSF with increasing volume of blood resulted in falsely elevated CSF protein concentrations. Hence, the amount of blood contamination has to be taken into consideration when interpreting CSF protein measurement results.

Project description:The analysis of cerebrospinal fluid (CSF) is used in biomarker discovery studies for various neurodegenerative central nervous system (CNS) disorders. However, little is known about variation of CSF proteins and metabolites between patients without neurological disorders. A baseline for a large number of CSF compounds appears to be lacking. To analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples of patients undergoing routine, non-neurological surgical procedures, we determined the variation of various proteins and metabolites by multiple analytical platforms. A total of 126 common proteins were assessed for biological variations between individuals by ESI-Orbitrap. A large spread in inter-individual variation was observed (relative standard deviations [RSDs] ranged from 18 to 148%) for proteins with both high abundance and low abundance. Technical variation was between 15 and 30% for all 126 proteins. Metabolomics analysis was performed by means of GC-MS and nuclear magnetic resonance (NMR) imaging and amino acids were specifically analyzed by LC-MS/MS, resulting in the detection of more than 100 metabolites. The variation in the metabolome appears to be much more limited compared with the proteome: the observed RSDs ranged from 12 to 70%. Technical variation was less than 20% for almost all metabolites. Consequently, an understanding of the biological variation of proteins and metabolites in CSF of neurologically normal individuals appears to be essential for reliable interpretation of biomarker discovery studies for CNS disorders because such results may be influenced by natural inter-individual variations. Therefore, proteins and metabolites with high variation between individuals ought to be assessed with caution as candidate biomarkers because at least part of the difference observed between the diseased individuals and the controls will not be caused by the disease, but rather by the natural biological variation between individuals.

Dataset Information

Ethanol contamination of cerebrospinal fluid during standardized sampling and its effect on (1)H-NMR metabolomics.

Publications

Ethanol contamination of cerebrospinal fluid during standardized sampling and its effect on (1)H-NMR metabolomics.

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