title: International validation of pyrogen tests based on cryopreserved human primary blood cells.
abstract: Pyrogens as fever-inducing agents can be a major health hazard in parenterally applied drugs. For the control of these contaminants, pyrogen testing for batch release is required by pharmacopoeias. This has been done either by the in vivo rabbit pyrogen test (since 1942) or the limulus amoebocyte lysate test (LAL), since 1976. New approaches include cell-based assays employing in vitro culture of human immune cells which respond e.g. by cytokine production (IL-1beta; IL-6) upon contact with pyrogens. Six variants of these assays have been validated in a collaborative international study. The recent successful development of cryopreservation methods promises to make standardized immunoreactive primary human blood cells available for widespread use. Furthermore, the pretesting of donors for infectious agents such as HIV or hepatitis has made it possible to develop a safe and standardised reagent for pyrogen testing. Using a total of 13 drugs, we have validated the pyrogen test based on fresh and cryopreserved human whole blood in four laboratories. The test reached >90% sensitivity and specificity. In contrast to the LAL, the test was capable of detecting non-endotoxin pyrogens derived from Gram-positive bacteria or fungi.
authors.affiliation: Biochemical Pharmacology, University of Konstanz, Universitätsstr.10, D-78457 Konstanz, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Schindler
authors.forename: Ingo
authors.initials: I
authors.lastname: Spreitzer
authors.forename: Bettina
authors.initials: B
authors.lastname: Löschner
authors.forename: Sebastian
authors.initials: S
authors.lastname: Hoffmann
authors.forename: Kilian
authors.initials: K
authors.lastname: Hennes
authors.forename: Marlies
authors.initials: M
authors.lastname: Halder
authors.forename: Peter
authors.initials: P
authors.lastname: Brügger
authors.forename: Esther
authors.initials: E
authors.lastname: Frey
authors.forename: Thomas
authors.initials: T
authors.lastname: Hartung
authors.forename: Thomas
authors.initials: T
authors.lastname: Montag
mesh_terms: D000826:Animal Testing Alternatives; D001769:Blood; D015925:Cryopreservation; D006801:Humans; D008070:Lipopolysaccharides / Q000032:analysis / Q000097:blood*; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000097:blood*; D011749:Pyrogens / Q000032:analysis / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D008070:Lipopolysaccharides; D004364:Pharmaceutical Preparations; D011749:Pyrogens