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Assessment of pyrogenic contaminations with validated human whole-blood assay

Nature Protocols volume 4, pages 1709–1721 (2009)Cite this article

Abstract

We present an internationally validated protocol for the evaluation of pyrogenic contaminations using human whole blood. In the in vitro pyrogen test (IPT) the sample is incubated with fresh or cryopreserved human whole blood, and the proinflammatory cytokine interleukin-1β (IL-1β) is detected by enzyme-linked immunosorbent assay (ELISA). In addition to detecting pyrogenic contaminations in aqueous samples, e.g., parenteral drugs; adaptations allow the assessment of lipidic, toxic or immunomodulatory substances; detection of low-grade contaminations in large-volume parenterals, e.g., dialysis water and fluids; pyrogenicity assessment of solid materials, e.g., medical devices; and evaluation of airborne pyrogenic burden. In contrast to the rabbit pyrogen test and the limulus amoebocyte lysate (LAL) test, it requires no components of animal origin. In comparison with the LAL, it also detects nonlipopolysaccharide pyrogens. In comparison with other monocyte activation tests it requires no cell preparation steps or cell culture facilities. The procedure takes 21–35 h to complete.

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Figure 1
Figure 2: Illustration of an enzyme-linked immunosorbent assay (ELISA) plate after the completion of an in vitro pyrogen test (IPT).
Figure 3: Analysis of pyrogenic activity of a cooling lubricant sample.
Figure 4: Air quality assessment with the in vitro pyrogen test (IPT).
Figure 5: Assessment of pyrogenic activity or interference of a steel sample by the in vitro pyrogen test (IPT).
Figure 6: Comparison of the response of fresh and cryopreserved human whole blood to different concentrations of lipopolysaccharides (LPS).

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Acknowledgements

The authors acknowledge the contributions of countless scientists, technicians, students, authorities and funding institutions to the development and validation of these protocols.

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Authors and Affiliations

  1. Biochemical Pharmacology, University of Konstanz, Konstanz, Germany

    Mardas Daneshian, Sonja von Aulock & Thomas Hartung

  2. Zukunftskolleg, University of Konstanz, Konstanz, Germany

    Sonja von Aulock

  3. Doerenkamp–Zbinden-Chair for Evidence-based Toxicology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA

    Thomas Hartung

Authors
  1. Mardas Daneshian
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  3. Thomas Hartung
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All authors contributed to the development of the method and the writing of the manuscript.

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Correspondence to Thomas Hartung.

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Daneshian, M., von Aulock, S. & Hartung, T. Assessment of pyrogenic contaminations with validated human whole-blood assay. Nat Protoc 4, 1709–1721 (2009). https://doi.org/10.1038/nprot.2009.159

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