Standard operating procedures for pre-analytical handling of blood and urine for metabolomic studies and biobanks.
Author(s): Bernini P, Bertini I, Luchinat C, Nincheri P, Staderini S, Turano P
Publication: J Biomol NMR, 2011, Vol. 49, Page 231-43
PubMed ID: 21380509 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of centrifugation speed (450 x g, 1000 x g, 3000 x g, or 11000 x g), filtering or adding sodium azide to centrifuged or uncentrifuged specimens, frozen storage temperature, and storage of preserved or unpreserved specimens at -80 degrees C and subsequently at room temperature on NMR profiles. Urine from 2 individuals was aliquoted, centrifuged for 5 min at 4 degrees C or left uncentrifuged, subjected to various pretreatments (filtration, enzymatic inhibitors, sodium azide or none), frozen for 1 week or used fresh, and stored for up to 24 h at room temperature.
Summary of Findings:
The difference between the first component of the spectra from the centrifuged and uncentrifuged specimens was greatest when the centrifugation speed was 1,000 or 3,000 relative centrifugal force (RCF). NMR spectra from specimens centrifuged at 11,000 RCF were closer to those in the specimens that were uncentrifuged, which the authors attribute to breaking down of the cells. The effect of centrifugation speeds was more pronounced in urine with a high cellular content. When urine was centrifuged prior to frozen storage for a week, the NMR profile was different from that when specimens were not centrifuged prior to frozen storage, but the magnitude of the difference was greater when the specimens were stored at -80 degrees C rather than in liquid nitrogen (8% as much change) or when NMR profiles from fresh centrifuged and uncentrifuged urine were compared (9% as much change). Importantly, changes observed after freezing or centrifugation were very small compared to the interindividual variability. Fresh or previously frozen, but unpreserved urine stored for 24 h at room temperature showed a spectral shift in pH sensitive metabolites as the specimen became more alkaline. Importantly, with storage at room temperature, succinate and acetate increased while urea, lactate, and glutamate decreased. However, specimens that were centrifuged and filtered showed fewer changes in spectra with storage at room temperature than specimens that were preserved with sodium azide, those that were unpreserved, or those only centrifuged prior to analysis or storage at -80 degrees C. When urine was stored for 24 h in an inert atmosphere rather than the normal atmosphere, the only difference was a slight reduction in the change in succinate. Addition of acetohydroxamic acid inhibited the decrease in urea with storage, and addition of EDTA at least partially attenuated the change in succinate and urea with storage.
Biospecimens
Preservative Types
- Frozen
- Other Preservative
- None (Fresh)
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Small molecule NMR Carbohydrate NMR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Filtration Filtered
Unfiltered
Storage Time at room temperature 0 h
2 h
4 h
6 h
24 h
Storage Storage conditions Inert atmosphere
Normal atmosphere
Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)
Sodium azide
Biospecimen Preservation Cooling or freezing method/ rate Liquid nitrogen
Direct transfer to freezer
Biospecimen Preservation Enzyme inhibitor Acetohydroxamic acid
EDTA
None
Biospecimen Aliquots and Components Cell number High cellular content
Low cellular content
Biospecimen Aliquots and Components Centrifugation Multiple speeds compared
Not centrifuged
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Study Purpose
The purpose of this study was to determine the effects of anticoagulant type, storage prior to or after centrifugation and storage temperature, duration, and conditions on NMR profiles from blood.
Summary of Findings:
Storage of whole blood from 10 individuals in EDTA or serum tubes prior to centrifugation caused increasing alterations in the NMR profiles with increasing storage duration. The changes in NMR spectra were more pronounced in specimens stored at 25 degrees C compared to specimens stored at 4 degrees C. Specifically, the authors identified significant decreases in glucose, particularly in serum, and an increase in lactate. The authors report that anticoagulant type had no effect on the changes in glucose, lactate or pyruvate in plasma with storage at 4 or 25 degrees C. When plasma or serum from 5 individuals was stored at room temperature after centrifugation, the changes in the NMR profiles were more pronounced for serum than for plasma. The shift in the NMR profiles with storage after centrifugation was driven by decreases in triglycerides, proline, choline, albumin, histidine, and low density lipoprotein/very low density lipoproteins (LDL/VLDL) and an increase in citrate. The histidine changes corresponded to those expected with a pH increase of 0.1. The authors report that the effect on LDL/VLDL was attenuated when specimens were stored in an inert atmosphere rather than a normal atmosphere and that storage of citrated or EDTA plasma had no effects on proline levels, but proline decreased with storage in fluoride/oxalate plasma. There was no difference in NMR profiles between serum stored in the light or dark at room temperature.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Small molecule NMR Carbohydrate NMR Lipid NMR Lipoprotein NMR Steroid NMR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage conditions Normal atmosphere
Inert atmosphere
Light
Dark
Storage Storage duration 0 h
1 h
2 h
3 h
4 h
6 h
12 h
18 h
24 h
Storage Storage temperature 4 degrees C
25 degrees C
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Whole blood
Biospecimen Acquisition Anticoagulant EDTA
Fluoride-oxalate
Sodium citrate
None
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated