Rapid potency assessment of autologous peripheral blood stem cells by intracellular flow cytometry: the PBSC-IL-3-pSTAT5 assay |
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| Institution: | 1. Medical Affairs and Innovation, Héma-Québec, Québec City, Canada;2. Cord Blood Bank, Héma-Québec, Montréal, Canada;3. Centre for Innovation, Canadian Blood Services, Ottawa, Canada;4. Biochemistry, Microbiology and Immunology Department, University of Ottawa, Ottawa, Canada;1. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA;2. Duke University, Durham, NC, 27708, USA;3. Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, 94305, USA;4. Center for Definitive and Curative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA;1. Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, Canada;2. School of Epidemiology and Public Health, University of Ottawa, Ottawa, Canada;3. Patient Partner, Ottawa Hospital Research Institute, Ottawa, Canada;4. Department of Anesthesiology and Pain Management, University of Toronto, Toronto, Canada;5. Department of Physiology, University of Toronto, Toronto, Canada;6. Population Health Research Institute, Hamilton Health Sciences, Hamilton, Canada;7. Department of Anesthesiology and Pain Medicine, University of Ottawa, Ottawa, Canada;8. Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Canada;9. Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada;10. School of Psychology, University of Ottawa, Ottawa, Canada;1. Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China;2. Immunotherapy Research Center for Hematologic Diseases of Hubei Province, Wuhan, Hubei, China;1. Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada;2. Centre for Journalology, Clinical Epidemiology Program, The Ottawa Hospital Research Institute, Ottawa, Canada;3. School of Epidemiology and Public Health, Faculty of Medicine, University of Ottawa, Ottawa, Canada;4. Department of Anesthesiology and Pain Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Canada;5. Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada;6. Neonatology, Department of Pediatrics, Children''s Hospital of Eastern Ontario (CHEO) and CHEO Research Institute, Ottawa, ON, Canada |
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| Abstract: | Background aimsThe current gold standard for stem cell product potency assessment, the colony-forming unit (CFU) assay, delivers results that are difficult to standardize and requires a substantial amount of time (up to 14 days) for cellular growth. Recently, the authors developed a rapid (<24 h) flow cytometry assay based on the measurement of intracellular phosphorylated STAT5 (pSTAT5) in CD34+ cord blood stem and progenitor cells in response to IL-3 stimulation. The present work presents a novel adaptation of the protocol for use with autologous peripheral blood stem cells (PBSCs) and a performance comparison with the CFU assay.MethodsThe flow cytometry intracellular staining assay was optimized for PBSCs, and patient samples were analyzed using the PBSC-IL-3-pSTAT5 and CFU assays. Warming events were also simulated to emulate impaired potency products.ResultsOptimization led to minor protocol adjustments, such as removal of the red blood cell lysis step, the addition of a formaldehyde fixation step and an increase in anticoagulant concentration. The PBSC-IL-3-pSTAT5 assay discriminated between normal and impaired samples and identified 100% (18 of 18) of the impaired samples, thus showing better specificity than the CFU assay.ConclusionsThe updated IL-3-pSTAT5 potency assay has several important advantages, such as accelerating the release of autologous stem cell products and enabling the detection of potentially impaired products. The assay could also be used to rapidly assess the potency of any cryopreserved allogeneic stem cell product, such as those processed during the coronavirus disease 2019 pandemic. |
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