Elsevier

Clinical Biochemistry

Volume 65, March 2019, Pages 38-44
Clinical Biochemistry

Impact of clinical sample handling and processing on ultra-low level measurements of plasma cytokines

https://doi.org/10.1016/j.clinbiochem.2019.01.001Get rights and content

Abstract

Objectives

In this study, we evaluated the impact of clinical sample handling and processing on IL-6, IL-10, IFNγ, and IL-2 measurements in plasma.

Design and methods

We collected whole blood samples and analyzed various pre-analytical parameters. We assessed the following: 1) cytokine stability in whole blood that was stored over a ten-hour period at room temperature and 4 °C; 2) cytokine stability in plasma over 6 h; 3) vigorous sample handling including repeated dropping and transport through a pneumatic transport system; and 4) freeze-thaw stability of cytokines in plasma. To ensure ability to measure IL-6, IL-10, IFNγ, and IL-2 levels in plasma, we used Simoa, an ultra-sensitive immunoassay platform.

Results

We show that whole blood storage at room temperature results in decreased cytokine levels and that whole blood storage at 4 °C results in greater cytokine stability. We also show that cytokines are stable when whole blood samples are subjected to vigorous sample handling. Lastly, we show that cytokines are stable in plasma over three freeze-thaw cycles.

Conclusions

Clinical sample handling and processing can affect measurements of IL-6, IL-10, IFNγ, and IL-2 in plasma. We believe this study will be a useful reference for future studies in which these cytokines are used as potential biomarkers.

Introduction

Cytokines are important immune signaling molecules that are associated with various diseases including cancer, neurological disorders, and autoimmune diseases [1]. Cytokines can exert their function at the local tissue level but can also be released and enter peripheral circulation. Changes in cytokine expression levels in blood have been shown to be biologically significant, making them attractive biomarker candidates for diagnosing disease, monitoring response to therapy, and monitoring disease progression. For example, cytokines can be used as part of a blood biomarker panel to assess rheumatoid arthritis disease activity [2]. Yet, despite the vast literature implicating cytokines with disease, the clinical utility of cytokines is still very limited.

Cytokine measurements in biological fluids are challenging for several reasons. First, cytokines vary in concentration between different individuals, sometimes by several orders of magnitude [3]. Second, blood cytokine levels do not necessarily correlate to local tissue expression levels due to the presence of soluble cytokine receptors, antibodies, and other factors that bind to cytokines and interfere with their detection. For example, IL-2 binding to its solubilized receptor, IL-2R, is known to interfere with immunoassays and may therefore lead to underestimated IL-2 levels [4]. Third, many cytokines are present at very low levels in the blood, and are difficult to measure using conventional techniques. Thus, due to limitations in analytical measurements of cytokines and difficulties in establishing cut-offs for healthy and disease states, the use of cytokines in disease diagnosis has been limited.

In addition to the challenges described above, variations in sample handling and processing in the clinical laboratory prior to analytical cytokine measurements can complicate accurate quantification [5]. Particularly, at ultra-low cytokine levels, small changes can have major consequences for reliable quantification. Downstream processing of whole blood can also affect cytokine measurements. For example, prior to plasma separation from whole blood, leukocytes can secrete cytokines in vitro and alter plasma levels [6]. Additionally, platelet activation can be accompanied by cytokine release, potentially resulting in apparently elevated cytokine levels in plasma that are not indicative of the underlying clinical condition [7]. Cytokine stability may also affect reliable quantification. Some cytokines have particularly short-half-lives, are temperature labile, and are subject to proteolytic degradation [8]. Plasma has been shown to have fewer matrix effects than serum when measuring a wide range of cytokines [9]. Additionally, heparin, which is used to process whole blood to plasma, has been associated with endotoxin contamination, which can induce cytokine release from monocytes [10]. Therefore, whole blood processing and handling can affect cytokine measurements and obscure disease associations.

In this study, we evaluated the effects of clinical specimen handling and processing on measurements of IL-6, IL-10, IFNγ, and IL-2 in plasma. IL-6, IL-10, IFNγ, and IL-2 are often present at ultra-low levels in blood [11,12]. To ensure our ability to quantify low cytokine levels, we used an ultra-sensitive assay technology known as Single Molecule Arrays (Simoa) [13,14]. Simoa assays have been previously demonstrated for ultra-sensitive detection of proteins and nucleic acids [[15], [16], [17], [18]] and their low limits of detection are accompanied by low CVs, which was important for this study. We used this method to study the stability of these cytokines under different clinical handling and processing conditions. We assessed various parameters including cytokine stability in whole blood that was stored over a ten-hour period at room temperature and 4 °C, cytokine stability in plasma over 6 h, vigorous sample handling including repeated dropping and transport through a pneumatic transport system, and freeze-thaw stability of cytokines in plasma. We show that in some cases, pre-analytical sample handling and processing can contribute to measurement variations. We conclude that it is important to consider pre-analytical parameters when measuring cytokines in blood for biomarker studies.

Section snippets

Simoa assay description

Simoa assays are bead-based immunoassays with the major advance of signal detection by single molecule counting, which results in ultra-high sensitivity. Antibody-coated capture beads are added in large excess to a sample containing low concentrations of target analyte molecules. Poisson statistics dictate that either one or zero target protein molecules will bind to each bead. The beads are then incubated with a biotinylated detection antibody and streptavidin-β-galactosidase, forming an

Cytokine stability in whole blood and plasma

We first assessed the variability in plasma cytokine measurements when whole blood was stored over a period of 2, 6, or 10 h. Whole blood contains white blood cells that can secrete cytokines, which may result in increased cytokine levels. On the other hand, lymphocyte-mediated internalization or binding to lymphocyte-derived soluble receptors may result in decreased cytokine levels [21]. Cytokine degradation, unfolding, or instability in whole blood may also result in decreased cytokine

Conclusions

In this study, we assessed various clinical sample handling and processing parameters on IL-6, IL-10, IFNγ, and IL-2 measurements in plasma. We show that storage of whole blood at room temperature over a period of hours can result in decreased cytokine levels. We also show that whole blood storage at 4 °C results in greater cytokine stability. Physical processes, such as dropping and pneumatic transport, as well as freeze-thawing do not seem to affect cytokine levels. While the measurements

Conflict of interest

The authors declare the following competing financial interest: David R. Walt is the scientific founder and a board member of Quanterix Corporation. All other authors declare no competing financial interest.

Acknowledgements

LC and DRW were funded by DARPA (HR0011-12-2-0001; Pass-through-entity: Univ. of North Carolina Chapel-Hill, subaward 5055065). AK and SEFM were supported by the BWH Department of Pathology. We thank the volunteers who donated their blood for stability studies.

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