title: Quality control of extracorporeal photochemotherapy: Proliferation assay using CFSE validated according to ISO 15189:2007 standards.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Faculté des sciences pharmaceutiques et biologiques, Université Paris-Descartes, Paris, France; Etablissement Français du Sang, Unité d'Ingénierie et de Thérapie Cellulaire, Créteil, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Faivre
authors.forename: Lucie
authors.initials: L
authors.lastname: Lecouflet
authors.forename: Wang-Qing
authors.initials: WQ
authors.lastname: Liu
authors.forename: Isabelle
authors.initials: I
authors.lastname: Khadher
authors.forename: Camille
authors.initials: C
authors.lastname: Lahaie
authors.forename: Michel
authors.initials: M
authors.lastname: Vidal
authors.forename: Sabine
authors.initials: S
authors.lastname: Legouvello
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Beaumont
authors.forename: Philippe
authors.initials: P
authors.lastname: Bierling
authors.forename: Hélène
authors.initials: H
authors.lastname: Rouard
authors.forename: Brigitte
authors.initials: B
authors.lastname: Birebent
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods / Q000592:standards*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006086:Graft vs Host Disease / Q000473:pathology / Q000628:therapy*; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects / Q000473:pathology / Q000528:radiation effects*; D017893:Photopheresis / Q000379:methods / Q000592:standards*; D010835:Phytohemagglutinins / Q000494:pharmacology; D017410:Practice Guidelines as Topic; D011786:Quality Control; D012751:Sezary Syndrome / Q000473:pathology / Q000628:therapy*; D013194:Staining and Labeling / Q000379:methods; D014466:Ultraviolet Rays
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D010835:Phytohemagglutinins
keywords: CFSE; biological assay; lymphocyte proliferation; photochemotherapy; quality control