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

van der Sar SA, Zielman R, Terwindt GM, van den Maagdenberg AM, Deelder AM, Mayboroda OA, Meissner A, Ferrari MD - Anal Bioanal Chem (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.

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.

No MeSH data available.


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In-house sample processing protocol used for preparation of samples for mass spectrometry (MS)-based metabolomics (methods 1 and 2) and for 1H-NMR-based metabolomics (method 3). All aliquots were immediately placed on dry ice within 30 min of sampling and then transferred to −80 °C for storage within 60 min
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Fig1: In-house sample processing protocol used for preparation of samples for mass spectrometry (MS)-based metabolomics (methods 1 and 2) and for 1H-NMR-based metabolomics (method 3). All aliquots were immediately placed on dry ice within 30 min of sampling and then transferred to −80 °C for storage within 60 min

Mentions: The cohort in which we initially discovered ethanol contamination consisted of human CSF samples that were collected for research purposes, namely to investigate the pathophysiology of migraine. The study protocol was approved by the Medical Ethical Committee of Leiden University Medical Center (LUMC), and all subjects gave written informed consent prior to collection. For disinfection of the skin, chlorhexidine (5 g/L)/denatured ethanol 70 % (Pharmacy LUMC, art. no.: 909602) was used. CSF samples were taken via lumbar puncture. Three different sample handling protocols were used simultaneously for the preparation of samples for mass spectrometry (MS)-based metabolomics (methods 1 and 2) and for 1H-NMR-based metabolomics (method 3) (see Fig. 1). Method 3 was used for the initial 1H-NMR measurements in which we detected ethanol contamination. For sampling of method 3, 4.8 mL of CSF dripped through air into a 15-mL polypropylene falcon tube. Directly after sampling, the CSF was centrifuged at 4 °C for 5 min (2000 rpm, 747 g). Following centrifugation, the supernatant was transferred to a 15-mL polypropylene falcon tube and divided in 0.5-mL aliquots, placed on dry ice within 30 min of sampling and transferred to −80 °C for storage within 60 min of sampling. In sampling methods 1 and 2, cold ethanol was added to the CSF during the sample handling to denature proteins and to be able to thaw the samples before measurements at lower temperatures. Because this was a likely source of ethanol contamination, we also analysed three CSF samples obtained for clinical diagnostic purposes to check whether ethanol is also present in these samples. Disinfection of the skin with chlorhexidine (5 g/L)/denatured ethanol 70 %, sample collection and sample processing of these clinical samples were similar to the research samples with the exception that ethanol was not used anywhere in the handling of these samples. For additional details on sampling and processing of the research and clinical CSF samples, see the Electronic Supplementary Material (ESM).Fig. 1


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

van der Sar SA, Zielman R, Terwindt GM, van den Maagdenberg AM, Deelder AM, Mayboroda OA, Meissner A, Ferrari MD - Anal Bioanal Chem (2015)

In-house sample processing protocol used for preparation of samples for mass spectrometry (MS)-based metabolomics (methods 1 and 2) and for 1H-NMR-based metabolomics (method 3). All aliquots were immediately placed on dry ice within 30 min of sampling and then transferred to −80 °C for storage within 60 min
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4446525&req=5

Fig1: In-house sample processing protocol used for preparation of samples for mass spectrometry (MS)-based metabolomics (methods 1 and 2) and for 1H-NMR-based metabolomics (method 3). All aliquots were immediately placed on dry ice within 30 min of sampling and then transferred to −80 °C for storage within 60 min
Mentions: The cohort in which we initially discovered ethanol contamination consisted of human CSF samples that were collected for research purposes, namely to investigate the pathophysiology of migraine. The study protocol was approved by the Medical Ethical Committee of Leiden University Medical Center (LUMC), and all subjects gave written informed consent prior to collection. For disinfection of the skin, chlorhexidine (5 g/L)/denatured ethanol 70 % (Pharmacy LUMC, art. no.: 909602) was used. CSF samples were taken via lumbar puncture. Three different sample handling protocols were used simultaneously for the preparation of samples for mass spectrometry (MS)-based metabolomics (methods 1 and 2) and for 1H-NMR-based metabolomics (method 3) (see Fig. 1). Method 3 was used for the initial 1H-NMR measurements in which we detected ethanol contamination. For sampling of method 3, 4.8 mL of CSF dripped through air into a 15-mL polypropylene falcon tube. Directly after sampling, the CSF was centrifuged at 4 °C for 5 min (2000 rpm, 747 g). Following centrifugation, the supernatant was transferred to a 15-mL polypropylene falcon tube and divided in 0.5-mL aliquots, placed on dry ice within 30 min of sampling and transferred to −80 °C for storage within 60 min of sampling. In sampling methods 1 and 2, cold ethanol was added to the CSF during the sample handling to denature proteins and to be able to thaw the samples before measurements at lower temperatures. Because this was a likely source of ethanol contamination, we also analysed three CSF samples obtained for clinical diagnostic purposes to check whether ethanol is also present in these samples. Disinfection of the skin with chlorhexidine (5 g/L)/denatured ethanol 70 %, sample collection and sample processing of these clinical samples were similar to the research samples with the exception that ethanol was not used anywhere in the handling of these samples. For additional details on sampling and processing of the research and clinical CSF samples, see the Electronic Supplementary Material (ESM).Fig. 1

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.

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.

No MeSH data available.


Related in: MedlinePlus