Changes in circulating cytokine and chemokine levels have been associated with many human diseases, and thus understanding the relationships between these changes and disease is an important area of medical research. Circulating levels of these proteins or other chemistries are measured from plasma or serum collected from peripheral blood draws. It is important to note that the methods of sampling and storage of plasma or serum are critical for accurate measurements. Here are some important considerations when planning to measure the levels of cytokines and chemokines in serum or plasma.
Blood collection tubes are available in a choice of factors and should be selected based on the analysis being done, as different anti-coagulants support different chemistries. Plasma is collected from blood drawn into tubes containing anticoagulants, including sodium or lithium heparin which act to inhibit thrombin from blood clotting, or sodium citrate or EDTA which chelate calcium ions to prevent coagulation. Serum collection tubes contain clot activators, however this method does not allow collection of peripheral blood mononuclear cells (PBMCs) from the same vial, which means that oftentimes, plasma will be the product of choice to maximize the value of blood drawn in a minimal number of tubes from study participants and healthy donors.
Following collection, plasma or serum should be cryopreserved at -80º C. Cytokines and chemokine levels can be measured by Enzyme-linked immunosorbent assay (ELISA). However, this method is time consuming and allows measurement of only one factor at a time. Luminex, a bead-based multiplex assay, can measure up to 100 cytokines, chemokines, or other soluble proteins at a time. Thus, for a given disease cohort, multitudes of measurements can be made from a single small sample of serum or plasma. Notably, many cytokines and chemokines exist in very low levels in peripheral blood, thus for each cytokine or chemokine to be measured it is important to determine if the detection range of the assay used is sufficient for the known range of circulating levels of that protein. Also, levels of these proteins may differ depending on whether they were measured in serum or plasma collected in various anticoagulants, so determinations should be done using the most similar methodologies as comparisons.
A methodology paper by de Jager et. al, discusses several important considerations for analyzing cytokine levels from serum or plasma by Luminex. In this paper, due to unmeasurably low levels of many cytokines, to allow for more dynamic determinations, whole blood was spiked with recombinant cytokines, or treated with LPS for a time period to upregulate expression of cytokines, prior to plasma collection, cryopreservation, and Luminex assays.
One comparison made was the difference in profiles of 15 cytokines in serum, versus plasma from the same donors collected in sodium heparin, EDTA, or citrate. Overall, cytokine levels were similar with a few exceptions, including IL-6 having the lowest values in serum compared with plasma, while CXCL8 was significantly higher in serum. The authors concluded that plasma collected in sodium heparin allowed the best measurements overall for the cytokines assessed. The time it takes to process and store samples after blood collection may also influence cytokine levels and should be done as consistently as possible for the most robust comparisons.
Another hugely important factor is sample storage time. As with all assays, experimental variation should be minimized, and thus it is common to store plasma or serum samples until the entire cohort has been collected and then analyze all of the samples simultaneously. This also comes into play when changes in cytokine profiles over time are to be measured from serial samples from an individual. The authors measured cytokine levels from sodium heparin plasma stored at -80º C over time, for up to four years. Several cytokines including IL-13, IL-15, IL-17 and CXCL8 began to be degraded within one year of storage, while levels of IL-1α, IL-1β, IL-5, IL-6, and IL-10 were degraded by over 50% in 2-3 years. IL-2, IL-4, IL-12 and IL-18 were much more stable, maintaining their initial levels out to 3 years post initial storage. Thus, depending on the cytokines being analyzed it is critical to keep these issues in mind. These are the same issues that are faced with storage of recombinant proteins that are used to generate the ELISA or Luminex standard curves or in other cytokine assays.
Stability of cytokines following several rounds of freeze-thawing were also assessed. Almost all of the cytokines analyzed with the exception of IL-6 and IL-10 were affected by freeze thawing the samples. Thus, when storing plasma or serum samples, it is important to freeze the samples in multiple aliquots such that additional assays can be performed while avoiding this issue.
In conclusion, handling and storage of serum and plasma samples as well as the choice of serum versus plasma collected in different anti-coagulants are all important factors to consider when planning for studies that will include measurement of circulating cytokines and chemokines.
Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays. de Jager W, Bourcier K, Rijkers GT, Prakken BJ, Seyfert-Margolis V. BMC Immunol. 2009 Sep 28;10:52. doi: 10.1186/1471-2172-10-52.