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Current practices and prospects for standardization of the hematopoietic colony-forming unit assay: a report by the cellular therapy team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative
Authors:Derwood Pamphilon  Eileen Selogie  David McKenna  Jose A. Cancelas-Peres  Zbigniew M. Szczepiorkowski  Ron Sacher  John McMannis  Hermann Eichler  Henk Garritsen  Minoko Takanashi  Leo van de Watering  David Stroncek  Jo-Anna Reems
Affiliation:1. NHS Blood and Transplant North Bristol Park, Filton, UK;2. Universtiy of Minnesota, St Paul, Minnesota, USA;3. Hoxworth Blood Center, Cincinnati, Ohio, USA;4. Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA;5. Hoxworth Blood Center, Cincinnati, Ohio, USA;6. University of Texas, M.D. Anderson Cancer Center, Houston, Texas;7. Institute of Clinical Hemostaseology and Transfusion Medicine, Saarland University Hospital, Homburg/Saar, Germany;8. Staedtisches Klinikum Braunschweig gGMbH, Braunschweig, Germany;9. Japanese Red Cross Kanto-Koshinetsu block Blood Centre, Tokyo, Japan;10. LUMC Jon J. van Rood Center for Clinical Transfusion Research, Leiden, Netherlands;11. National Institutes of Health, Bethesda, Maryland, USA;12. Puget Sound Blood Center, Seattle, Washington, USA;13. University of Washington, Seattle, Washington, USA;14. ENet Answers, Manhattan Beach, CA, USA;1. Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA;2. Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA;1. Aikenhead Centre for Medical Discovery, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Melbourne, VIC 3010, Australia;2. Department of Politics, Media, and Philosophy, La Trobe University, Bundoora, VIC 3086, Australia;3. School of Humanities, University of Tasmania, Hobart, TAS, Australia;4. School of Humanities and Social Inquiry, University of Wollongong, Wollongong, NSW 2522, Australia;1. Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA;2. Waisman Biomanufacturing, University of Wisconsin-Madison, Madison, Wisconsin, USA;3. Department of Surgery, University of Wisconsin-Madison, Madison, Wisconsin, USA;4. Molecular & Cellular Therapeutics, University of Minnesota, Minneapolis, Minnesota, USA;5. Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital, Texas Children''s Hospital, Houston, Texas, USA;6. EMMES Corporation Inc, Rockville, Maryland, USA;7. University of Wisconsin Carbone Cancer Center, Madison, Wisconsin, USA
Abstract:Background aimsWide acceptance of the colony-forming unit (CFU) assay as a reliable potency test for stem cell products is hindered by poor inter-laboratory reproducibility. The goal of this study was to ascertain current laboratory practices for performing the CFU assay with an eye towards identifying practices that could be standardized to improve overall reproducibility.MethodsA survey to evaluate current laboratory practices for performing CFU assays was designed and internationally distributed.ResultsThere were 105 respondents to the survey, of whom 68% performed CFU assays. Most survey recipients specified that an automated rather than a manual cell count was performed on pre-diluted aliquots of stem cell products. Viability testing methods employed various stains, and when multiple sites used the same viability stain, the methods differed. Cell phenotype used to prepare working cell suspensions for inoculating the CFU assay differed among sites. Most respondents scored CFU assays at 14–16 days of incubation, but culture plates were read with various microscopes. Of 57 respondents, 42% had not performed a validation study or established assay linearity. Only 63% of laboratories had criteria for determining if a plate was overgrown with colonies.ConclusionsSurvey results revealed inconsistent inter-laboratory practices for performing the CFU assay. The relatively low number of centers with validated CFU assays raises concerns about assay accuracy and emphasizes a need to establish central standards. The survey results shed light on numerous steps of the methodology that could be targeted for standardization across laboratories.
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