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Variables that affect assays for plasma cytokines and soluble activation markers
N Aziz1, P Nishanian, R Mitsuyasu
1CIRID at University of California, Los Angeles, Department of Microbiology and Immunology, Los Angeles, California 90095, USA.
Insights
Assays for cytokines and immune markers in HIV patients show significant variability between manufacturers and lots, impacting diagnostic accuracy. Standardizing these tests is crucial for reliable clinical interpretation and longitudinal studies.
Area of Science:
- Clinical immunology
- Biomarker analysis
- Diagnostic assay development
Background:
- Cytokines and soluble immune activation markers are vital for diagnosing and understanding diseases like HIV.
- Measuring these markers in biological fluids offers prognostic and pathogenetic insights.
Purpose of the Study:
- To evaluate the performance of various assays for cytokines and immune activation markers.
- To identify factors influencing assay outcomes, particularly in HIV-positive and HIV-negative individuals.
Main Methods:
- Assessed neopterin, beta2-microglobulin, soluble interleukin-2 receptor, soluble tumor necrosis factor receptor type II, tumor necrosis factor alpha, and interferon-gamma (IFN-gamma).
- Utilized serum and plasma samples from HIV-positive and HIV-negative subjects.
- Compared results from enzyme-linked immunosorbent assay (ELISA) kits from different manufacturers and lots.
Main Results:
- Significant variability in analyte levels was observed between different ELISA kit manufacturers and even different lots from the same manufacturer (up to 50% difference).
- International reference standards exist for cytokines but not for soluble cytokine receptors or activation markers.
- Analyte levels in serum and plasma were generally similar, except for IFN-gamma; most analytes were stable under various storage conditions.
Conclusions:
- Standardization of assay kits and development of international reference standards are essential for accurate quantitative measurements.
- For longitudinal studies, consistent use of pre-verified reagents from a single manufacturer is recommended.
- Proficiency testing and external quality assurance programs are vital to ensure laboratory performance and clinical relevance.
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.
