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Quality control of extracorporeal photochemotherapy: Proliferation assay using CFSE validated according to ISO 15189:2007 standards
Author(s) -
Faivre Lionel,
Lecouflet Lucie,
Liu WangQing,
Khadher Isabelle,
Lahaie Camille,
Vidal Michel,
Legouvello Sabine,
Beaumont JeanLouis,
Bierling Philippe,
Rouard Hélène,
Birebent Brigitte
Publication year - 2015
Publication title -
cytometry part b: clinical cytometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 61
eISSN - 1552-4957
pISSN - 1552-4949
DOI - 10.1002/cyto.b.21188
Subject(s) - flow cytometry , microbiology and biotechnology , chemistry , chromatography , biology
Background For the last 40 years, the technique of extracorporeal photopheresis (ECP) has constantly developed. Among irradiation systems, those called “off‐line” allow the validation of the quality of the cell therapy product. The inhibition of the proliferation of lymphocytes after ultraviolet irradiation (UVA) is usually verified by the tritiated thymidine assay as in vitro proliferation assay. The document presented here describes the results obtained while performing the setting up of an alternative proliferation assay using flow cytometry according to ISO 15189:2007 Standard. Methods Cells samples taken before and after UVA irradiation were labeled with CarboxyFluorescein Succinimidyl Ester (CFSE) and then cultured with phytohemagglutinin‐A (PHA). After 3 days, an analysis of the CFSE staining was realized by flow cytometry. In order to validate the shift in the method used according to Standard, the following tests were performed: 1) comparison with the reference method, 2) robustness test, 3) reagents stability. Results Comparison method demonstrated that the sensitivity of the CFSE test is 100%, the specificity is 89%, and the concordance is almost complete. The CFSE test is robust regarding parameters like cell concentration or PHA concentration. PHA and CFSE are stable for 6 months and one year, respectively. Conclusion Validation of this alternative test, according to the ISO 15189:2007 Standard, has demonstrated good concordance with reference method. The results of the robustness and stability of reagents are appropriate for its routine use. Thus, the benefits of alternative technique make it a wise choice for the quality control of ECP in a cell therapy laboratory. © 2014 International Clinical Cytometry Society