[Evaluation and further development of a pyrogenicity assay based on human whole blood]

When cells of the immune system, i.e. primarily blood monocytes and macrophages, come into contact with pyrogens (fever inducing contaminations) they release mediators transmitting the fever reaction within the organism. A new pyrogen test exploits this reaction for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample. In case of pyrogen contamination, the formation of interleukin-1 is induced, which is determined by ELISA. According to the various pharmacopoeia, the rabbit pyrogen test determines the fever reaction following injection of a test sample. In comparison, the new whole blood assay is more sensitive, less expensive and determines the reaction of the targeted species. In contrast to the well established in vitro alternative, i.e. the limulus amebocyte lysate assay (LAL), the blood assay is not restricted to endotoxins of Gram-negative bacteria and is not to the same extent disturbed by endotoxin-binding blood proteins. Here, interim results of the ongoing optimisation and prevalidation are demonstrated. Preliminary data of the evaluation for biological and pharmaceutical drugs are presented.

[A new test for the detection of pyrogens in pharmaceutical products. Examinations for the validation of the human whole blood assay]

The human whole blood assay utilises the natural fever response to detect pyrogens by determination of the release of IL-1beta. In order to replace the official method, the rabbit pyrogen test, a validation of the whole blood assay is necessary. A comparison of the results obtained from many blood samples has revealed the following: 1) Blood not stimulated by LPS does not produce IL-1beta. 2) Stimulation by LPS induces a concentration-dependent release of IL-1( beginning at a concentration of between 2-5 pg/mL LPS. 3) The amount of IL-1beta released varies greatly between samples obtained from different individuals. 4) Storing blood samples results in a right shifted LPS/IL-1beta curve with a steeper gradient and higher maximum value of IL-1beta. In this paper we suggest an experimental method for the determination of pyrogens based on the established semi-quantitative LAL gelation method as detailed in the European Pharmacopeia. Using this methodology, we were able to show that the amount of endotoxin in a number of different infusion solutions was below the LAL-endotoxin limit concentration. LPS was quantitatively determined from spiked samples.