The importance of sample collection when using single cytokine levels and systemic cytokine profiles as biomarkers--a comparative study of serum versus plasma samples.
Author(s): Tvedt TH, Rye KP, Reikvam H, Brenner AK, Bruserud Ø
Publication: J Immunol Methods, 2015, Vol. 418, Page 19-28
PubMed ID: 25637409 PubMed Review Paper? No
Purpose of Paper
This paper compared levels of thirty-one cytokines, four soluble adhesion molecules, and eight matrix metalloproteinases (MMPs) in matched serum and EDTA, heparin, and sodium citrate plasma from healthy patients. The effect of collection into bivalirudin instead of citrate and addition of platelet inhibitors on levels of CXC chemokine ligand (CXCL) 5, vascular endothelial growth factor (VEGF), and MMP-9 was also examined.
Conclusion of Paper
Hierarchical clustering of soluble adhesion molecules, MMPs, and chemokine and cytokine levels in serum and the various plasma types separated specimens into two main clusters: all serum specimens plus one citrate plasma specimen and all remaining plasma specimens. The plasma specimens then divided into three clusters, two predominantly containing a mixture of matched EDTA and citric acid specimens and the remaining cluster containing 17 of 20 heparin specimens and the EDTA and citrate specimens from the same patient. Levels of epidermal growth factor (EGF), VEGF, CD40 ligand (CD40L), P-selectin, thrombopoietin (TPO), MMP-1, MMP-8, and MMP9 were higher in serum than plasma. Levels of CD40L, EGF, and MMP-1 tended to be lower in EDTA plasma than heparin or citrate plasma. CXCL10 and CXCL11 were highest in heparin plasma and levels of MMP-2 were highest in EDTA plasma. The coefficient of variance depended on the analyte and specimen type. Platelet counts were significantly correlated with levels of P-selectin in all specimen types and with sCD40, CXCL5, and EGF in some specimen types.
Levels of CXCL5 and MMP-9 were each higher for seven of eight specimens, and levels of VEGF were higher in four patients when plasma was collected using bivalirudin instead of citrate, but there was no significant effect from the addition of platelet inhibitors.
Studies
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Study Purpose
This study compared levels of thirty-one cytokines, four soluble adhesion molecules, and eight MMPs in matched serum and EDTA, heparin, and sodium citrate plasma from 20 healthy patients. Peripheral venous blood was collected from each patient into four different Vacutainer tubes: serum tube with clot activator, EDTA, heparin, and citrate. Serum tubes were allowed to clot for 2 h at room temperature before separation of the serum by centrifugation at 1300 x g for 10 min. Plasma tubes were centrifuged at 2000 x g for 15 min at room temperature within 30 min of venipuncture. Plasma and serum were aliquoted and stored at -80˚C. Levels of interleukin (IL)-1alpha, IL-1beta, IL-1 receptor antagonist (IL1-RA), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-17, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL5, CXCL10, CXCL11, basic fibroblast growth factor (bFGF), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), VEGF, TPO, EGF, hepatocyte growth factor (HGF), Leptin, interferon (IFN)-gamma, CD40L, tumor necrosis factor (TNF)alpha, MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, intracellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), E-selectin, and P-selectin were determined by Luminex immunoassays.
Summary of Findings:
Comparisons between specimen types found levels of IL1-RA and CCL3 were similar while levels of VCAM-1, ICAM-1, E-selectin, P-selectin, CCL2, TPO, Leptin, MMP-2, MMP-3, CCL2, CCL4, and MMP-1 levels were dependent on specimen type but still correlated between most combinations. Levels of CCL5, CCL11, CXCL5, CXCL11, CD40L, MMP-1, MMP-7, MMP-8, and MMP-9 differed among specimen types and were generally not correlated among specimen types. Hierarchical clustering of soluble adhesion molecules, MMPs, and chemokine and cytokine levels in serum and the various plasma types separated specimens into two main clusters with the lower group containing three subclusters. The upper cluster included all 20 serum specimens and one plasma specimen (citrate). Two of the lower clusters contained a mixture of EDTA and citric acid specimens (8 EDTA and 11 citrate in one, and 11 EDTA and 6 citrate in the other) with specimens from the same individual tending to cluster together (6 pairs and 5 pairs). The final group contained 17 of the 20 heparin specimens and one paired EDTA and citrate specimen. Levels of EGF, VEGF, CD40L, P-selectin, TPO, MMP-1, MMP-8, and MMP9 were higher in serum than plasma. Levels of CD40L, EGF, and MMP-1 tended to be lower in EDTA plasma than heparin or citrate plasma. CXCL10 and CXCL11 were highest in heparin plasma, with CXCL10 levels being higher in all plasma specimens than serum and CXCL11 levels being below the range of detection in other plasma types and very low in serum. MMP-2 levels were 2-fold higher in EDTA plasma than in other specimen types. The authors report that median leptin levels are highest in EDTA plasma, but this contradicts the data table. The coefficient of variance was lowest in serum for CD40L, IL-1RA, CCL5, CXCL5, CXCL8, CXCL10, E-selectin, P-selectin, MMP-2, MMP-7, and MMP-8; in heparin plasma for CCL2, CCL11, TPO, HGF, and VCAM-1; in EDTA plasma for Leptin, MMP-1, MMP-3, and MMP-9; and in citric acid plasma for ICAM-1. Platelet counts were significantly correlated with levels of P-selectin in all tubes types (r=0.484-0.583, P=0.007-0.031), sCD40 levels in heparin (r=0.498, P=0.03) or citrate (r=0.491, P=0.038) plasma, levels of CXCL5 in citrate plasma (r=0.589, P=0.006), and EGF levels in EDTA plasma (r=0.523, P=0.022). Levels of TNF-alpha, INF-gamma, bFGF, IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-17, MMP-12, and MMP-13 were low or undetectable in the majority of specimens, regardless of collection tube type.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Citrate
EDTA
Heparin
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Biospecimen Acquisition Type of collection container/solution Serum tube with clot activator
Anticoagulant tube
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Study Purpose
This study investigated the effects of anticoagulation with bivalirudin versus citrate and the addition of platelet inhibitory agents on measurement of CXCL5, VEGF, and MMP-9 in plasma. Venous blood from eight healthy individuals was collected into Vacuette tubes containing bivalirudin, citric acid alone, citric acid with sodium salicylic acid and prostaglandin E2 (platelet-inhibitory agents), or citric acid plus ticagrelor (a platelet inhibitor). Tubes were centrifuged at 2000 x g for 10 min and stored at 4˚C for less than 24 h before analysis of CXCL5, VEGF, and MMP-9 by ELISA.
Summary of Findings:
Levels of CXCL5 and MMP-9 were higher for seven of eight specimens, and levels of VEGF were higher in four patients when plasma was collected using bivalirudin instead of citrate. Addition of sodium salicylic acid and prostaglandin E2 or ticagrelor did not significantly affect levels of CXCL5, MMP-9, or VEGF; although VEGF was only detectable in specimens from two of eight patients.
Biospecimens
Preservative Types
- Other Preservative
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Bivalirudin
Citrate
ELISA Specific Targeted peptide/protein CXCL5
MMP-9
VEGF
Biospecimen Aliquots and Components Biospecimen components Citric acid alone
Citric acid with sodium salicylic acid and prostaglandin E2
Citric acid plus ticagrelor