Reproducibility of serum cytokines and growth factors
Introduction
Cytokines and growth factors are critical regulators of immune responses, inflammatory reactions, and the processes of angiogenesis, apoptosis and cell proliferation that play important roles in chronic diseases such as coronary heart disease, diabetes, and cancer [1], [2], [3], [4], [5].
When blood samples are collected after the onset of disease, as in retrospective case-control studies, measurements of cytokines are meaningless as predictors of disease risk because of the potential for “reverse causation,” i.e., the disease process may have altered prior cytokine levels. For both economic and practical reasons, in most prospective cohort studies only one blood sample is collected per study subject, and exposure-disease associations are usually based on a single measurement. In order for a measurement to be useful, it should reflect the long-term cytokine level for the individual and not mere short-term variability, so the temporal reproducibility of measurements is important to establish before conducting a study of cytokine-disease associations. The degree to which a single measurement reflects the long-term average biomarker level for an individual, relative to others, depends on the between-person variability of these levels over time relative to the within-person variability, as indexed by the intraclass correlation coefficient (ICC) [6].
Several previous studies have assessed the temporal reproducibility for selected cytokines and growth factors in serum [7], [8], [9], [10], [11], [12], [13], [14], [15], but these studies have had various limitations: small sample size, assessment of only short-term variability, assessment of only a small number of biomarkers, or use of poor measures of reproducibility. Recent advances in analytical methods such as the Luminex technology allow simultaneous measurement of multiple biomarkers reducing costs, labor, and sample volume requirements [16], which is particularly important for prospective cohorts with banked biological specimens. Two recent studies have used this method to evaluate reproducibility of adipokines and other obesity-related biomarkers, including some cytokines such as IL-1β, IL-6, IL-8, and TNF-α, as well as growth factors such as NGF and HGF [14], [15]. However, data on the reproducibility of other cytokines and growth factors measured using Luminex are practically absent.
In the present study, we assessed the reproducibility of a number of cytokines and growth factors in serum from annually drawn blood specimens in a subset of participants from the NYU Women’s Health Study (NYUWHS), a large prospective cohort. We measured 23 serum cytokines (IL-1α, IL-1β, IL-1RA, IL-2, IL-2Rα, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-10, IL-12p40, hsIL-12p70, IL-13, IL-15, IL-17, TNF-α, TNF-R1, TNF-R2, IFN-α, IFN-γ, sCD40L), nine growth factors (GMCSF, EGF, bFGF, GCSF, HGF, VEGF, TGF-α, NGF, EGFR), and CRP in samples collected over a 2-year period. The biomarkers for this study were selected on the basis of the functional role in inflammatory processes, as well as the availability of commercial kits. In addition, for eight of the biomarkers (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α, IFN-γ), both regular and high-sensitivity assays were performed, resulting in 41 measurements for each sample that were analyzed for the current report.
Section snippets
The New York University Women’s Health Study (NYUWHS)
Between March 1985 and June 1991, 14,274 women 35–65 years old were enrolled at a mammography screening center in New York City. The cohort was restricted to women who in the preceding 6 months were neither pregnant nor treated with hormones. After signing the informed consent at the time of enrollment, and at annual screening visits thereafter, subjects were asked to complete questionnaires on lifestyle and dietary factors and to provide 30 mL of non-fasting peripheral venous blood, drawn using
Characteristics of study subjects
The mean age of the 65 subjects was 50.8 years and 85% of them were of European descent (Table 1). The mean age at first blood donation for premenopausal and postmenopausal women was 43.3 and 57.2 years, respectively. The mean body mass index at time of the first blood donation for premenopausal and postmenopausal women was 23.9 and 24.2 kg/m2, respectively. Mean durations in storage of the serum samples from premenopausal women were 20.0, 18.8, and 17.5 years for visits 1, 2, and 3,
Discussion
We used Luminex xMap™ technology to investigate the ability of a single measurement of several cytokines or growth factors to represent a subject’s long-term average levels, relative to other subjects. To our knowledge, this is the first study to measure the temporal reproducibility of a large number of cytokines and growth factors using Luminex methodology. The results demonstrated that 16 out of the 41 biomarkers measured were quite stable in a woman over time, so that a single measurement of
Acknowledgments
The authors thank Lynne Quinones and Yelena Afanasyeva for technical assistance. This research was supported by the National Institutes of Health Grants R01 CA98661 and R03 CA96428, and by Cancer Center Grant CA16087 from the National Cancer Institute and the NIEHS Center Grant ES00260.
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