Effect of Delayed Centrifugation on the Levels of NMR-Measured Lipoproteins and Metabolites in Plasma and Serum Samples.
Author(s): Debik J, Isaksen SH, Strømmen M, Spraul M, Schäfer H, Bathen TF, Giskeødegård GF
Publication: Anal Chem, 2022, Vol. 94, Page 17003-17010
PubMed ID: 36454175 PubMed Review Paper? No
Purpose of Paper
This paper compared the metabolite profiles and lipoprotein subfractions of serum and plasma from healthy patients and obese patients obtained after a centrifugation delay of up to 8 h at room temperature. Additionally, serum and plasma profiles from the same individual were compared, and the authors explored whether the ratio of lactate to glucose could be used to identify specimens obtained after a centrifugation delay.
Conclusion of Paper
Principal component analysis (PCA) trajectory plots of plasma and serum metabolic profiles revealed that the largest shifts in the metabolic profiles of specimens were due to delayed centrifugation, with 25 of 33 metabolites in plasma affected and 25 of 32 metabolites in serum affected. In many cases, serum and plasma metabolites displayed similar changes in the affected metabolites, although glutamic acid and dimethyl-sulfone displayed an inverse response when serum and plasma were compared. Conversely, PCA based on lipoprotein fractions clustered specimens by patient rather than by centrifugation delay. In PCA based on the metabolite profile of serum or plasma, specimens from obese and healthy patients overlapped, but specimens from obese patients clustered separately from those from healthy patients when clustered based on the lipoprotein subfractions. Serum and plasma profiles from the same patient did not co-cluster in PCA analysis based on the metabolite profile, and 28 of the 32 metabolites quantified in both specimen types differed significantly between plasma and serum specimens. In contrast, PCA based on the lipoprotein subfraction co-clustered serum and plasma from the same individual patient. The ratio of lactate to glucose in plasma and serum increased linearly with the duration of pre-centrifugation storage. Based on the area under the receiver operating curve (ROC-AUC) for the ratio of lactate to glucose, plasma and serum specimens that experienced a delay to centrifugation ≥1 h had a ROC-AUC of 0.87 and 0.90, respectively.
Studies
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Study Purpose
This study compared the metabolite profiles of serum and plasma from healthy patients and obese patients obtained after a centrifugation delay of up to 8 h. Additionally, serum and plasma profiles from the same individual were compared, and the authors explored whether the ratio of lactate to glucose could be used to identify specimens obtained after a centrifugation delay. Blood was collected from 10 patients at an obesity clinic and 10 other donors (diagnosis not specified); blood was collected into EDTA-plasma vacuette tubes and serum vacutainer tubes with a clot activator. Blood was stored at room temperature for 30 min, 1, 2, 4, or 8 h before plasma and serum were separated by centrifugation (speed and duration not specified). Plasma and serum were stored at -80°C. Forty-one metabolite levels were quantified by nuclear magnetic resonance (NMR). Metabolites with levels below the limit of detection in ≥30% of specimens were omitted from analysis, reducing the number of metabolites to 33 in plasma and 32 in serum. The Benjamini Hochberg procedure was used to correct P-values for multiple testing, and significance was defined as a Q-values of 0.05.
Summary of Findings:
The coefficient of variance (CV) was below 15% and 20% for 23 and 27 of 33 plasma metabolites, respectively, and 22 and 25 of 32 serum metabolites, respectively. The plasma NMR spectra revealed decreases (glucose) and increases (lactic acid and glycine) in select metabolites and an increase in the K2EDTA peak after a 1 h delay to centrifugation. PCA trajectory plots of plasma and serum metabolic profiles revealed that the largest shifts in metabolic profiles of specimens were due to delayed centrifugation, with 25 of 33 plasma and 25 of 32 serum metabolites affected. After an 8 h delay to centrifugation, the largest decreases in plasma and serum were in methionine (-61% and -20%, respectively), acetic acid (-61% and -24%, respectively), and glucose (-25% and -21%, respectively). After an 8 h centrifugation delay, the metabolites with the largest increases in plasma and serum were lactic acid (232% and 164%, respectively), ornithine (147% in plasma, excluded from analysis in serum), and N-N-dimethylglycine (23% and 84%, respectively). While an 8 h delay to centrifugation led to decreased levels of glutamic acid (-61%) and increased dimethyl-sulfone concentration (24%) in plasma, the reverse occurred in serum, as serum glutamic acid increased (212%), and dimethyl-sulfone decreased (-35%). Importantly, centrifugation delays also led to increased mean CV in levels of metabolites in plasma (23.6% from 13.3%) and serum (22.2% from 17.7%). The ratio of lactate to glucose in plasma and serum increased linearly with the duration of pre-centrifugation storage. Based on the area under the receiver operating curve (ROC-AUC) for the ratio of lactate to glucose, plasma and serum specimens that experienced a delay to centrifugation ≥1 h had a ROC-AUC of 0.87 and 0.90, respectively and for a >2 h delay the ROC-AUC increased to 0.94 and 0.95, respectively. In PCA based on the metabolite profile of serum and plasma, specimens from obese and healthy patients overlapped and displayed similar effects of delayed centrifugation. However, plasma from obese patients had higher levels of creatinine, isoleucine, valine, and glucose and lower levels of glutamine, glycine, histidine, methionine, and acetic acid compared to plasma from healthy patients. In PCA analysis, the serum and plasma metabolite profiles from the same patient did not co-cluster and 28 of the 32 metabolites quantified in both serum and plasma differed significantly between plasma and serum.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Obesity
Platform:
Analyte Technology Platform Carbohydrate NMR Small molecule NMR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Obese
Healthy
Storage Storage duration 30 min
1 h
2 h
4 h
8 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
-
Study Purpose
This study compared the lipoprotein subfraction of serum and plasma from healthy patients and obese patients obtained after a centrifugation delay of up to 8 h. Additionally, serum and plasma profiles from the same individual were compared. Blood was collected from 10 patients in an obesity clinic and 10 other donors (diagnosis not specified); blood was collected into EDTA-plasma vacuette tubes and serum vacutainer tubes with a clot activator. Blood was stored at room temperature for 30 min, 1, 2, 4, or 8 h before plasma and serum were separated by centrifugation (speed and duration not specified). Plasma and serum were stored at -80°C. One hundred lipoprotein subfractions were quantified by NMR. The Benjamini Hochberg procedure was used to correct P-values for multiple testing, and significance was defined as a Q-values of 0.05.
Summary of Findings:
Principal component analysis (PCA) based on lipoprotein fractions clustered specimens by patient rather than by the centrifugation delay. Although, delayed centrifugation led to statistically significant changes in 78 lipoprotein subfractions in plasma and 74 lipoprotein subfractions in serum, changes >20% were only observed for 4 of these in plasma (21.6% for HDL-4 free cholesterol; -21.5% for HDL-3 free cholesterol; 20.9% for HDL-1 apo-A1 concentrations and 26.8% for free cholesterol in VLDL-5) and 9 of these in serum (-42.6% for LDL-2 free cholesterol; -39.2% for LDL-2 cholesterol;-34.8% for LDL-2 apo-B; -30.2% for LDL-2 phospholipids; 22.5% for VLDL cholesterol; 34.0% for LDL-4 triglycerides; 30.6% LDL-5 triglycerides; 33.6% for Intermediate-density lipoprotein free cholesterol; and 62.4% for Intermediate-density lipoprotein total cholesterol). Even with delayed centrifugation, the CVs in lipoprotein subfractions remained low in plasma (2.75% baseline versus 5.2% after 8 h delay) and serum (3.3% at baseline and 6.3% after 8 h delay). Not surprisingly, PCA based on lipoprotein fractions in plasma and serum clustered specimens from obese patients separately from those of healthy patients. Serum and plasma collected from obese patients had higher levels of triglycerides and VLDLs and lower levels of HDLs than specimens from healthy patients, but the stability of these analytes did not differ between obese and healthy patients. In PCA based on lipoprotein fractions, serum and plasma from individual patients co-clustered. Although levels of 91 of the 100 lipoprotein subfractions differed significantly between plasma and serum specimens, differences were <±20% for all but five of the subfractions.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Obesity
Platform:
Analyte Technology Platform Lipid NMR Steroid NMR Lipoprotein NMR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Obese
Healthy
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage duration 30 min
1 h
2 h
4 h
8 h
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum