TY - JOUR
T1 - Cryopreservation of human whole blood for pyrogenicity testing
AU - Schindler, Stefanie
AU - Asmus, Silvia
AU - Von Aulock, Sonja
AU - Wendel, Albrecht
AU - Hartung, Thomas
AU - Fennrich, Stefan
N1 - Funding Information:
This work was supported by the Bundesministerium für Bildung und Forschung (BMBF 11425A) and the Stiftung zur Förderung der Erforschung von Ersatz- und Ergänzungsmethoden zur Einschränkung von Tierversuchen (set). The procedure has been granted a European patent (97 122 072.8).
PY - 2004/11
Y1 - 2004/11
N2 - Human whole blood assays are increasingly employed to test immune function or detect pyrogenic contamination, since they offer advantages, such as ease of performance, few preparation artifacts and a physiological cell environment. However, the approach is often limited by the availability of freshly drawn blood, putative safety concerns in the case of infected donors and interindividual donor differences. To overcome these limitations, a method was developed and optimized to produce batches of cryopreserved blood that can be used directly after thawing without any washing steps. Mononuclear cells remained intact as shown by FACS analysis. Cytokine release could be induced by a variety of immunological stimuli. The cell preparation released higher amounts of interleukin-1β (IL-1β) and IL-6 compared to fresh blood, but no TNF. These differences could be attributed to the presence of the cryoprotectant dimethylsulfoxide (DMSO) alone by addition of DMSO to fresh blood. Large batches of cryopreserved blood could be produced by mixing blood donations of up to 10 donors, independent of differing blood groups. The detection limit for the World Health Organization (WHO) lipopolysaccharides (endotoxin, LPS) reference preparation (EC-6) with regard to the induction of IL-1β release was at least 0.5 endotoxin equivalent units (EU)/ml. Endotoxin spikes at the limit concentrations prescribed in the European Pharmacopoeia could be detected in a series of drugs, showing that the In vitro Pyrogen Test (IPT) can also be run with cryopreserved blood. Further possible applications include high-throughput screening for immunomodulators or toxins as well as preservation of patient samples for later analysis of cell functions.
AB - Human whole blood assays are increasingly employed to test immune function or detect pyrogenic contamination, since they offer advantages, such as ease of performance, few preparation artifacts and a physiological cell environment. However, the approach is often limited by the availability of freshly drawn blood, putative safety concerns in the case of infected donors and interindividual donor differences. To overcome these limitations, a method was developed and optimized to produce batches of cryopreserved blood that can be used directly after thawing without any washing steps. Mononuclear cells remained intact as shown by FACS analysis. Cytokine release could be induced by a variety of immunological stimuli. The cell preparation released higher amounts of interleukin-1β (IL-1β) and IL-6 compared to fresh blood, but no TNF. These differences could be attributed to the presence of the cryoprotectant dimethylsulfoxide (DMSO) alone by addition of DMSO to fresh blood. Large batches of cryopreserved blood could be produced by mixing blood donations of up to 10 donors, independent of differing blood groups. The detection limit for the World Health Organization (WHO) lipopolysaccharides (endotoxin, LPS) reference preparation (EC-6) with regard to the induction of IL-1β release was at least 0.5 endotoxin equivalent units (EU)/ml. Endotoxin spikes at the limit concentrations prescribed in the European Pharmacopoeia could be detected in a series of drugs, showing that the In vitro Pyrogen Test (IPT) can also be run with cryopreserved blood. Further possible applications include high-throughput screening for immunomodulators or toxins as well as preservation of patient samples for later analysis of cell functions.
KW - Blood
KW - Cryopreservation
KW - Endotoxin
KW - In vitro Pyrogen Test (IPT)
KW - Interleukin-1β
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U2 - 10.1016/j.jim.2004.08.019
DO - 10.1016/j.jim.2004.08.019
M3 - Article
C2 - 15604019
AN - SCOPUS:10344243492
SN - 0022-1759
VL - 294
SP - 89
EP - 100
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -