Preanalytical aspects and sample quality assessment in metabolomics studies of human blood.
Author(s): Yin P, Peter A, Franken H, Zhao X, Neukamm SS, Rosenbaum L, Lucio M, Zell A, Häring HU, Xu G, Lehmann R
Publication: Clin Chem, 2013, Vol. 59, Page 833-45
PubMed ID: 23386698 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to assess potential impacts of anticoagulant use and type on chemical noise in during mass spectroscopy analyses. S-monovette collection tubes containing 4 different anticoagulants (lithium heparinate, sodium fluoride, sodium citrate, potassium EDTA) or kaolin (serum) were used for blood collection. Blood specimens from three volunteers were evaluated by mass spectroscopy for chemical noise [(-CH2CH2O-)n]. Controls were completed for each tube type investigated and contained saline with 50 g/L human albumin. Plasma specimens were gently mixed and stored in ice water for 25 min, while serum specimens were permitted to clot for 25 min at room temperature prior to centrifugation.
Summary of Findings:
Chemical noise inference was clearly detected as mass spectroscopy signals in blood specimens collected in tubes containing plastic beads, which included lithium heparinate and serum collection tubes. Collection tubes with the least amount chemical noise compared to control tubes were potassium EDTA and sodium fluoride collection tubes.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Small molecule LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Sodium citrate
Sodium fluoride
Potassium EDTA
Lithium heparin
None
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Study Purpose
This purpose of this study was to investigate the influence of varying degrees of sample hemolysis on clinical chemistry and metabolomic analyses in potassium-EDTA blood specimens collected from 10 volunteers. Hemolysis was induced by aspirating EDTA blood specimens through a 26-gauge cannula once or twice, while controls were centrifuged at 4 degrees C immediately after collection.
Summary of Findings:
The following clinical chemistry analytes were significant elevated in moderate and strong hemolytic specimens compared to controls (p<0.05): free hemoglobin, aspartate transamine, and potassium. Conversely, C reactive protein levels were equivalent among samples regardless of the degree of hemolysis. Ultraperformance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (qTOF-MS) using a nontargeted approach revealed that hemolysis elicited pronounced effects on the metabolomic profile of plasma. A PCA score plot, revealed greater variability among the hemolytic specimens compared to controls, and a heat map showed 69 features that differed significantly between hemolytic and control specimens (p<0.05). Specific metabolites identified by the authors that differed between hemolytic and control specimens included lysophosphatidylcholines(lyso-P) C16:0 and C18:0 (p<0.05).
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Protein Clinical chemistry/auto analyzer Electrolyte/Metal Clinical chemistry/auto analyzer Small molecule LC-TOF-MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Hemolysis Absent
Fine needle aspiration-induced
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Study Purpose
The purpose of this study was to determine if the duration and temperature of a processing delay affects the metabolomic composition of EDTA blood specimens. Blood from 10 volunteers was collected in potassium EDTA tubes and stored at room temperature for 0, 2, 4, 8, or 24 h or chilled in ice water for 2 or 4 h. Portions of the experiment was repeated with blood collected from an additional 5 (4 and 24 h delay at room temperature) or 10 (1 and 4 h delay in ice water) volunteers to analyze the kinetics of the hypoxanthine and S-1-P peak areas.
Summary of Findings:
The authors note that a room temperature delay in processing resulted in a general decrease in signal intensity. As shown on the PCA score plot, specimens subjected to a room temperature delay also displayed greater variability compared to immediately processed controls. A total of 64 features were identified on the heat map that showed significant differences in the peak area between specimens subjected to a room temperature delay of 2 to 24 h compared to immediately processed controls (p<0.05). Three of the most altered features were later identified as the metabolites hypoxanthine, sphingosine 1-phosphate (S-1-P), and linolenyl carnitine (p<0.05 for all). The peak area of hypoxanthine increased by 800% after a 2 h delay at room temperature compared to 0 h controls (p=0.02), increasing by of 4010% after a 24 h delay (p=0.002). The peak area of S-1-P increased 380% after a 2 h delay at room temperature compared to 0 h controls (p=0.02), and 1250% after a 24 h delay (p=0.002). The concentration for linolenyl carnitine did not differ from 0 h controls until 8 h at room temperature at which time a 1780% increase in peak area occurred compared to 0 h controls (p=0.016), after which levels plateaued (p=0.008). Conversely, storage of specimens in ice water for 2 or 4 h did not result in any obvious changes in the plasma metabolome when compared to immediately processed controls. Although data was not shown, the authors note that all 64 features altered by room temperature storage displayed no alterations when specimens were stored in ice water for 2 or 4 h.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Small molecule LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0, 2, 4, 8, 24 h
Storage Storage temperature Room temperature
Ice water
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
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Study Purpose
The purpose of this study was to determine if the metabolomic profile of EDTA plasma specimens is susceptible to freeze-thaw cycling. Blood collected from 10 volunteers was collected into potassium EDTA tubes and processed immediately. Plasma specimens were frozen at -80 degrees C and thawed in ice water a total of 1, 2, or 4 times. Results were compared to the data collected from case-matched controls that were not frozen.
Summary of Findings:
A low level of variability was observed between fresh plasma specimens and those subjected to a single thaw event on wet ice. A total of 4 features were significantly different between plasma specimens subjected to 2 freeze-thaw events and fresh specimens (p<0.05). One of the features was identified as L-carnitine, which exhibited a 70% decline in peak area after 2 or 4 freeze-thaw events compared to fresh controls (p<0.05 for both).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Small molecule LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
4 cycles