Validation of a flow cytometry-based method to quantify viable lymphocyte subtypes in fresh and cryopreserved hematopoietic cellular products |
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| Authors: | Bechara Mfarrej Julie Gaude Jerome Couquiaud Boris Calmels Christian Chabannon Claude Lemarie |
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| Affiliation: | 1. Department of Chemistry and Biotechnology, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Australia;2. Regenerative Medicine and Stem Cell Laboratory, Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Medak, India;3. ARC Training Centre in Surface Engineering for Advanced Materials, School of Engineering, Swinburne University of Technology, Hawthorn, Australia;1. Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam;2. Vinmec HiTech Center, Vinmec Healthcare System, Hanoi, Vietnam;3. College of Health Science, VinUniversity, Hanoi, Vietnam;1. Anthony Nolan Cell Therapy Centre, Nottingham, UK;2. The Anthony Nolan Research Institute, Nottingham, UK;1. Department of Regenerative Technologies and Biofabrication, National Medical Research Radiological Center, Obninsk, Russia;2. Basel University, Basel, Switzerland;1. Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, Maryland, USA;2. University Health Network, Toronto, Canada;3. University of Toronto, Toronto, Canada;4. Akron Biotech, Boca Raton, Florida, USA;5. Massachusetts Institute of Technology Center for Biomedical Innovation, Boston, Massachusetts, USA;6. Frost and Sullivan, San Francisco, California, USA;7. Georgia Institute of Technology, Atlanta, Georgia, USA;8. Cell & Gene Therapy Catapult, London, UK;9. BioFabUSA, Manchester, New Hampshire, USA;10. Centre for Commercialization of Regenerative Medicine, Toronto, Canada;11. Novartis, Cambridge, Massachusetts, USA;12. Standards Coordinating Body for Gene, Cell, and Regenerative Medicines and Cell-Based Drug Discovery, Gaithersburg, Maryland, USA;1. Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy;2. Sezione di Ematologia, Dipartimento di Scienze Radiologiche ed Ematologiche, Università Cattolica del Sacro Cuore, Rome, Italy |
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| Abstract: | Background aimsAdoptive cellular therapy with immune effector cells (IECs) has shown promising efficacy against some neoplastic diseases as well as potential in immune regulation. Both inherent variability in starting material and variations in cell composition produced by the manufacturing process must be thoroughly evaluated with a validated method established to quantify viable lymphocyte subtypes. Currently, commercialized immunophenotyping methods determine cell viability with significant errors in thawed products since they do not include any viability staining. We hereby report on the validation of a flow cytometry-based method for quantifying viable lymphocyte immunophenotypes in fresh and cryopreserved hematopoietic cellular products.MethodsUsing fresh or frozen cellular products and stabilized blood, we report on the validation parameters accuracy, uncertainty, precision, sensitivity, robustness and contamination between samples for quantification of viable CD3+, CD4+ T cells, CD8+ T cells, CD3–CD56+CD16+/– NK cells, CD19+ B cells and CD14+ monocytes of relevance to fresh and cryopreserved hematopoietic cellular products using the Cytomics FC500 cytometer (Beckman Coulter).ResultsThe acceptance criteria set in the validation plan were all met. The method is able to accommodate the variability in absolute numbers of cells in starting materials collected or cryopreserved from patients or healthy donors (uncertainty of ≤20% at three different concentrations), stability over time (compliance over 3 years during regular inter-laboratory comparisons) and confidence in meaningful changes during cell processing and manufacturing (intra-assay and intermediate precision of 10% coefficient of variation). Furthermore, the method can accurately report on the efficacy of cell depletion since the lower limit of quantification was established (CD3+, CD4+ and CD8+ cells at 9, 8 and 8 cells/µL, respectively). The method complies with Foundation for the Accreditation of Cellular Therapy (FACT) standards for IEC, FACT-Joint Accreditation Committee of ISCT-EBMT (JACIE) hematopoietic cell therapy standards, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q2(R1) and International Organization for Standardization 15189 standards. Furthermore, it complies with Ligand Binding Assay Bioanalytical Focus Group/American Association of Pharmaceutical Scientists, International Council for Standardization of Hematology/International Clinical Cytometry Society and European Bioanalysis Forum recommendations for validating such methods.ConclusionsThe implications of this effort include standardization of viable cell immunophenotyping of starting material for cell manufacturing, cell selection and in-process quality controls or dosing of IECs. This method also complies with all relevant standards, particularly FACT-JACIE standards, in terms of enumerating and reporting on the viability of the “clinically relevant cell populations.” |
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