Effect of anticoagulants on multiplexed measurement of cytokine/chemokines in healthy subjects

Abstract

Introduction Cytokines are humoral regulatory molecules that act together in immunologic pathways. Monitoring cytokines and their variations within physiologic ranges is critical for biomarker discovery. Therefore, we evaluated the performance characteristics of 72 analytes measured by multiplex cytokine immunoassay, with an emphasis on the differences of analytes measured in serum compared to plasma, and, in plasma, on the impact of anticoagulants on the cytokine measurement. Methods We used fluorescent bead-based (Luminex) immunoassay kits to simultaneously measure 72 analytes. We tested serum and plasma samples from 11 matched donors. Plasma samples were anti-coagulated with sodium heparin, sodium citrate dextrose and ethylene diamine tetra-acetic acid (EDTA), respectively. Results Of the 72 cytokines, 12 were undetectable in all types of specimen samples. Nineteen analytes, including PDGF-bb, IL-4, IL-8, IL-9, FGF-b, PAI-1, CXCL-5, CCL-5, CD40L, EGF, VEGF, IL-2ra, IL-3, SDF-1a, PCT, MCP-3, GIP, IL-16 and fibrinogen, showed significant differences between measurements in serum and all types of plasma, regardless of anticoagulant. Among plasma samples, 10 analytes (eotaxin, SCGF-b, MCP-1, SCF, MIP-1b, VEGF, RANTES, PDGF-b, PAI-1 and ITAC) showed significantly higher concentrations in heparinized plasma compared to citrated and EDTA plasma. IP-10, and CTAK were the only 2 cytokines that presented different concentrations in citrate and EDTA plasma. Conclusions With their small volume, low cost per test, and multiplex capacity, Luminex-based cytokine assays have enormous potential utility for screening in epidemiologic studies. In our study, we showed that many cytokines’ concentrations differed between serum and plasma samples, and that different anticoagulants used in preparation of plasma samples also affected the measurement of some cytokines. There was no optimal sample preparation that was clearly superior for the measurement of all analytes measured. Ultimately, the utility of cytokine measurement, as biomarker or to monitor the immune system, will depend on attention to detail in the collection and processing of samples in addition to assay precision.

Inverse association between maternal 25OHD level and cord GLP-1/GIP concentrations

“…In contrast to adults, nutrition is basically supplied through the placenta to fetuses, not through eating. Moreover, GLP-1 seems to be higher than in healthy adults using the same Bio-Rad suspension array system (10,34). However, the active form of GLP-1 in cord blood was reported to be close to the lower limit of detection (35).…”

“…However, this technique is not considered optimal for measuring levels of gut hormones (36). With other methods, levels of some of these hormones might differ markedly (10,34). We therefore compared diabetesrelated hormone levels as measured by BioPlex within samples of this study population, and not with results from other studies.…”

“…Third, we compared hormone levels in cord blood with those in healthy adults as provided by Bio-Rad Laboratories. However, the number of samples used for their data was only 10 or 11 (10,34). We can therefore make only a rough estimate of whether hormone levels seem higher in cord blood than in adults.…”

Proteomics to predict the response to tumour necrosis factor-α inhibitors in rheumatoid arthritis using a supervised cluster-analysis based protein score

“…The coagulation cascade is a complex biochemical process which involves activation of platelets and other cellular components, resulting in the release of various proteins. Therefore, the difference between serum and plasma in proteomic profile typically involves plateletrelated markers, such as IL-6, CCL5, CXCL4, CXCL8, and vascular endothelial growth factor A (29)(30)(31)(32). Of these, only IL-6 was selected as a potential marker.…”

Prognostic impact of preoperative serum interleukin‑6 levels in patients with early‑stage oral squamous cell carcinoma, defined by sentinel node biopsy

“…Sera derived from patients with early stage OSCC were previously examined for multiple cytokines using a multiplexed measurement system (14) and serum interand serum interleukin-6 (IL-6) level was revealed to negatively correlate with a favourable outcome in these patients. IL-6 is a multifunctional cytokine that functions in the regulation of inflammatory and immune responses.…”