Human plasma stability during handling and storage: impact on NMR metabolomics.
Author(s): Pinto J, Domingues MR, Galhano E, Pita C, Almeida Mdo C, Carreira IM, Gil AM
Publication: Analyst, 2014, Vol. 139, Page 1168-77
PubMed ID: 24443722 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of anticoagulant type, storage, freeze-thaw cycling and patient fasting on the plasma metabolome as determined by nuclear magnetic resonance (NMR).
Conclusion of Paper
Generally differences in the NMR spectra between specimens collected in EDTA and heparin tubes were less than the interindividual variability, but EDTA plasma specimens had higher levels of pyruvate and two unassigned compounds than heparinated plasma. When plasma was stored at room temperature, low density lipoprotein (LDL) and very LDL (VLDL) methylene (CH2) and methyl (CH3) groups decreased, and high density lipoprotein (HDL) methyl groups, phosphatidylcholine (PC) lyso-PC (LPC) and sphingomyelin (SM) increased after as little as 2 h. When specimens were stored at -20°C, increases in proline and glucose and a decrease in an unassigned peak were noted after 31 days. After 31 days at -80°C, a 50% decrease in the unassigned peak was observed. The authors report that storing specimens at -80°C for 6-12 months versus 14-19 months had no effects, but they report that specimens stored at -80°C for 20-30 months had 2% higher cholesterol and slight changes in N-acetyl glycorproteins and creatinine compared to specimens stored for 14-19 months. The effects of freeze-thaw cycling of plasma depended on the specimen, with higher lipid, lactate and choline content leading to greater degradation, and most changes appearing after 4 cycles. The authors report that the effect of fasting versus non-fasting depended on the individual, and most profiles largely overlapped.
Studies
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Study Purpose
The purpose of this study was to determine the effects of anticoagulant type, storage, freeze-thaw cycling and patient fasting on the plasma metabolome evaluated by NMR. Blood was collected from healthy pregnant and non-pregnant women into sodium heparin tubes (and EDTA tubes for anticoagulant study) and centrifuged within 30 min. Plasma was frozen at -20°C and then stored at -20°C or -80°C. Specimens were thawed for 30 min at room temperature. Plasma from 5 non-pregnant women was used for the anticoagulant study. Room temperature stability and freeze-thaw cycling were investigated using plasma from 3 pregnant women, and short-term storage at -20°C or -80°C was investigated in the plasma from 3 non-pregnant women. Long-term storage for 6-12, 14-19, and 20-30 months was investigated using plasma from 11, 17, and 21 pregnant women, respectively. The effect of fasting was investigated using the plasma from 16 non-pregnant women.
Summary of Findings:
Generally differences in the NMR spectra between specimens collected in EDTA and Heparin tubes were less than the interindividual variability, but EDTA plasma specimens had higher levels of pyruvate and two unassigned compounds than heparinated plasma (p=0.0023, p=0.0079, and p=0.0043, respectively). When plasma was stored at room temperature, LDL and VLDL CH2 and CH3 groups decreased, and HDL methyl groups, PC, LPC and SM increased after as little as 2 h, regardless of whether sodium azide was added. When specimens were stored at -20°C, a 31% increase in proline (p=0.033), a 9% increase in glucose (p=0.019) and a 50% decrease in an unassigned peak were noted after 31 days at -20°C (p=0.00036) or -80°C (p=0.0022). The authors report that storing specimens at -80°C for 6-12 months versus 14-19 months had no effects, but they report that specimens stored at -80°C for 20-30 months had 2% higher cholesterol and slight changes in N-acetyl glycoproteins and creatinine compared to specimens stored for 14-19 months. The effects of freeze-thaw cycling depended on the specimen, with the specimen containing higher lipid, lactate and choline content showing greater degradation, and most changes appearing after 4 cycles. With 5 freeze-thaw cycles, this particular specimen showed a decrease in lipids (5%) and acetone (16%) and 2-7% increases in choline phiospholipids, alanine, glucose and pyruvate. The authors report that the effect of fasting versus non-fasting depended on the individual, and most profiles largely overlapped.
Biospecimens
Preservative Types
- Frozen
- Other Preservative
- None (Fresh)
Diagnoses:
- Normal
- Pregnant
Platform:
Analyte Technology Platform Steroid NMR Carbohydrate NMR Lipid NMR Small molecule NMR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Heparin
EDTA
Storage Storage temperature -20°C
-80°C
Storage Time at room temperature 1 h
2 h
3.5 h
8.5 h
16.5 h
21 h
Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
4 cycles
5 cycles
Storage Storage duration 3 days
1 week
1 month
6-12 months
14-19 months
20-30 months
Preaquisition Patient diet Fasting
After eating
Biospecimen Preservation Type of fixation/preservation Sodium azide
None (fresh)