Haehnel, Viola and Dormann, Frauke and Kronenberg, Katharina and Hutchinson, James A. and Burkhardt, Ralph and Ahrens, Norbert (2021) Validation of an apoptosis assay for extracorporeal photopheresis. TRANSFUSION MEDICINE, 31 (2). pp. 113-120. ISSN 0958-7578, 1365-3148
Full text not available from this repository. (Request a copy)Abstract
Objectives This validation study investigated a flow cytometric apoptosis assay according to good manufacturing practice (GMP). Background Extracorporeal photopheresis (ECP) is a treatment for various immunological diseases and cutaneous T-cell lymphomas. It is based on the induction of apoptosis by 8-methoxypsoralene and ultraviolet A light. The quantification of apoptosis is therefore essential for ECP improvements. However, despite numerous publications on apoptosis, validated technical details are lacking. Methods and materials Mononuclear cells were collected by apheresis and treated by ECP or camptothecin. Samples taken before and after ECP were cultured for 24, 48 and 72 h and analysed for apoptosis and viability of T cells and monocytes by flow cytometry with Annexin V and 7-AAD staining. Accuracy of the assay, intra- and inter-assay precision and the pre-analytical and analytical stability of the analytes were the investigated parameters. Results Our data indicate that the median intra- and inter-assay precision coefficient of variation for T cells was 3.86% and 4.80%, respectively. Pre-analytical stability of T cells and monocytes was ensured during short-term storage for up to 2 h on ice. After staining, analytical stability was limited to 30 min, likely because of ongoing apoptosis and loss of monocytes due to plastic adhesion. Conclusion The results of this validation study show that the assay is GMP-compliant and that its reliability, accuracy and precision are acceptable. While pre-analytical stability of the cells was compatible with on-site procedures, our analytical stability data indicate that this assay is not suited for batch mode analysis of ECP products.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | ; apoptosis; ECP; extracorporeal photopheresis; validation |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Medicine > Lehrstuhl für Chirurgie Medicine > Lehrstuhl für Klinische Chemie und Laboratoriumsmedizin |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 09 Aug 2022 07:00 |
| Last Modified: | 09 Aug 2022 07:00 |
| URI: | https://pred.uni-regensburg.de/id/eprint/46168 |
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