Speeding up pyrogenicity testing: Identification of suitable cell components and readout parameters for an accelerated monocyte activation test (MAT).
Pyrogen testing represents a crucial safety measure for parental drugs and medical devices, especially in direct contact with blood or liquor. The European Pharmacopoeia regulates these quality control measures for parenterals. Since 2010, the monocyte activation test (MAT) has been an accepted pyrogen test that can be performed with different human monocytic cell sources: whole blood, isolated monocytic cells or monocytic cell lines with IL1β, IL6, or TNFα as readout cytokines. In the present study, we examined the three different cell sources and cytokine readout parameters with the scope of accelerating the assay time. We could show that despite all cell types being able to detect pyrogens, primary cells were more sensitive than the monocytic cell line. Quantitative real-time PCR revealed IL6 mRNA transcripts having the largest change in Ct-values upon LPS-stimulation compared to IL1β and TNFα, but quantification was unreliable. IL6 protein secretion from whole blood or peripheral blood mononuclear cells (PBMC) was also best suited for an accelerated assay with a larger linear range and higher signal-to-noise ratios upon LPS-stimulation. The unique combination with propan-2-ol or a temperature increase could additionally increase the cytokine production for earlier detection in PBMC. The increased incubation temperature could finally increase not only responses to lipopolysaccharides (LPS) but also other pyrogens by up to 13-fold. Therefore, pyrogen detection can be accelerated considerably by using isolated primary blood cells with an increased incubation temperature and IL6 as readout. These results could expedite assay time and thus help to promote further acceptance of the MAT. Copyright © 2016 John Wiley & Sons, Ltd.
University Hospital Tuebingen, Clinic for Thoracic, Cardiac and Vascular Surgery, Calwerstr. 7/1, 72076, Tuebingen, Germany.
Sandra
S
Stoppelkamp
University Hospital Tuebingen, Clinic for Thoracic, Cardiac and Vascular Surgery, Calwerstr. 7/1, 72076, Tuebingen, Germany.
Noriana
N
Würschum
University Hospital Tuebingen, Clinic for Thoracic, Cardiac and Vascular Surgery, Calwerstr. 7/1, 72076, Tuebingen, Germany.
Katharina
K
Stang
University Hospital Tuebingen, Clinic for Thoracic, Cardiac and Vascular Surgery, Calwerstr. 7/1, 72076, Tuebingen, Germany.
Jasmin
J
Löder
University Hospital Tuebingen, Clinic for Thoracic, Cardiac and Vascular Surgery, Calwerstr. 7/1, 72076, Tuebingen, Germany.
Meltem
M
Avci-Adali
University Hospital Tuebingen, Clinic for Thoracic, Cardiac and Vascular Surgery, Calwerstr. 7/1, 72076, Tuebingen, Germany.
Leila
L
Toliashvili
University Hospital Tuebingen, Clinic for Thoracic, Cardiac and Vascular Surgery, Calwerstr. 7/1, 72076, Tuebingen, Germany.
Christian
C
Schlensak
University Hospital Tuebingen, Clinic for Thoracic, Cardiac and Vascular Surgery, Calwerstr. 7/1, 72076, Tuebingen, Germany.
Hans Peter
HP
Wendel
University Hospital Tuebingen, Clinic for Thoracic, Cardiac and Vascular Surgery, Calwerstr. 7/1, 72076, Tuebingen, Germany.
Stefan
S
Fennrich
D002460:Cell Line; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008070:Lipopolysaccharides / Q000276:immunology; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D011749:Pyrogens / Q000032:analysis* / Q000276:immunology*; D012333:RNA, Messenger / Q000235:genetics
D016207:Cytokines; D008070:Lipopolysaccharides; D011749:Pyrogens; D012333:RNA, Messenger
endotoxins; medical devices; monocyte activation test; parenterals; pyrogens