Variables that affect assays for plasma cytokines and soluble activation markers

Abstract

Cytokines and soluble immune activation markers that reflect cytokine activities in vivo are increasingly being measured in plasma, serum, and other body fluids. They provide useful diagnostic and prognostic information as well as insight into disease pathogenesis. Assays of neopterin, beta2-microglobulin, soluble interleukin-2 receptor, and soluble tumor necrosis factor receptor type II as well as of the cytokines tumor necrosis factor alpha and gamma interferon (IFN-gamma) were evaluated by using serum and plasma samples of human immunodeficiency virus (HIV)-positive and HIV-negative subjects. Many factors were found to influence the outcomes of these assays. Substantial differences in apparent levels of analytes were frequently found when enzyme-linked immunosorbent assay (ELISA) kits from different manufacturers were used. In some cases, differences were found in the standards provided by separate manufacturers. Furthermore, the analytic results from different lots of ELISA kits supplied by single manufacturers differed by as much as 50%. The need for uniformity in the standards for quantitative assays was clearly illustrated. International reference standards are available for cytokines but not for soluble cytokine receptors or soluble activation markers. Marker levels in serum or in plasma were similar except those for IFN-gamma. Most of the analytes were stable under several storage conditions. Thus, batch testing of frozen stored samples is feasible. The findings indicate that for longitudinal studies, the levels of cytokines and immune activation markers in plasma or serum should be measured by using preverified reagents from one manufacturer. The quality of laboratory performance can have an impact on clinical relevance. Proficiency testing and external quality assurance programs can help to develop the needed consensus.

FIG. 1

Effects of repeated freezing and thawing on plasma levels of neopterin, β2-microglobulin, TNF-α, sTNF-RII, and sIL-2R. The data are the means ± standard errors for seven subjects (four healthy donors and three HIV-seronegative subjects) for each analyte.

FIG. 2

Influence of storage temperature on plasma levels of β2M, sIL-2R, neopterin, IFN-γ, sTNF-RII, and TNF-α. Box plots are shown for the analyte levels in serum and plasma samples from 11 subjects (6 HIV-seropositive patients and 5 HIV-seronegative donors) that were stored for 20 days at room temperature (24°C), refrigerated (4°C), and frozen (−70°C). For TNF-α and IFN-γ, the data are for the five donors and for five of the HIV-seropositive subjects. The line inside each box represents the median; the lower and upper edge of each box indicate the 25th and 75th percentiles, respectively; the vertical lines begin and end at the 10th and 90th percentile points; and the lower and upper circles indicate the 5th and 95th percentile points, respectively.