Setting objective thresholds for rare event detection in flow cytometry.
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2014-07
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The accurate identification of rare antigen-specific cytokine positive cells from peripheral blood mononuclear cells (PBMC) after antigenic stimulation in an intracellular staining (ICS) flow cytometry assay is challenging, as cytokine positive events may be fairly diffusely distributed and lack an obvious separation from the negative population. Traditionally, the approach by flow operators has been to manually set a positivity threshold to partition events into cytokine-positive and cytokine-negative. This approach suffers from subjectivity and inconsistency across different flow operators. The use of statistical clustering methods does not remove the need to find an objective threshold between between positive and negative events since consistent identification of rare event subsets is highly challenging for automated algorithms, especially when there is distributional overlap between the positive and negative events ("smear"). We present a new approach, based on the Fβ measure, that is similar to manual thresholding in providing a hard cutoff, but has the advantage of being determined objectively. The performance of this algorithm is compared with results obtained by expert visual gating. Several ICS data sets from the External Quality Assurance Program Oversight Laboratory (EQAPOL) proficiency program were used to make the comparisons. We first show that visually determined thresholds are difficult to reproduce and pose a problem when comparing results across operators or laboratories, as well as problems that occur with the use of commonly employed clustering algorithms. In contrast, a single parameterization for the Fβ method performs consistently across different centers, samples, and instruments because it optimizes the precision/recall tradeoff by using both negative and positive controls.
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Richards, Adam J, Janet Staats, Jennifer Enzor, Katherine McKinnon, Jacob Frelinger, Thomas N Denny, Kent J Weinhold, Cliburn Chan, et al. (2014). Setting objective thresholds for rare event detection in flow cytometry. J Immunol Methods, 409. pp. 54–61. 10.1016/j.jim.2014.04.002 Retrieved from https://hdl.handle.net/10161/14686.
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Thomas Norton Denny
Thomas N. Denny, MSc, M.Phil, is the Chief Operating Officer of the Duke Human Vaccine Institute (DHVI), Associate Dean for Duke Research and Discovery @RTP, and a Professor of Medicine in the Department of Medicine at Duke University Medical Center. He is also an Affiliate Member of the Duke Global Health Institute. Previously, he served on the Health Sector Advisory Council of the Duke University Fuquay School of Business. Prior to joining Duke, he was an Associate Professor of Pathology, Laboratory Medicine and Pediatrics, Associate Professor of Preventive Medicine and Community Health and Assistant Dean for Research in Health Policy at the New Jersey Medical School, Newark, New Jersey. He has served on numerous committees for the NIH over the last two decades and currently is the principal investigator of an NIH portfolio in excess of 65 million dollars. Mr. Denny was a 2002-2003 Robert Wood Johnson Foundation Health Policy Fellow at the Institute of Medicine of the National Academies (IOM). As a fellow, he served on the US Senate Health, Education, Labor and Pensions Committee with legislation/policy responsibilities in global AIDS, bioterrorism, clinical trials/human subject protection and vaccine related-issues.
As the Chief Operating Officer of the DHVI, Mr. Denny has senior oversight of the DHVI research portfolio and the units/teams that support the DHVI mission. He has extensive international experience and previously was a consultant to the U.S. Centers for Disease Control and Prevention (CDC) for the President’s Emergency Plan for AIDS Relief (PEPFAR) project to oversee the development of an HIV and Public Health Center of Excellence laboratory network in Guyana. In September 2004, the IOM appointed him as a consultant to their Board on Global Health Committee studying the options for overseas placement of U.S. health professionals and the development of an assessment plan for activities related to the 2003 PEPFAR legislative act. In the 1980s, Mr. Denny helped establish a small laboratory in the Republic of Kalmykia (former Soviet Union) to improve the care of children with HIV/AIDS and served as a Board Member of the Children of Chernobyl Relief Fund Foundation. In 2005, Mr. Denny was named a consulting medical/scientific officer to the WHO Global AIDS Program in Geneva. He has also served as program reviewers for the governments of the Netherlands and South Africa as well as an advisor to several U.S. biotech companies. He currently serves as the Chair of the Scientific Advisory Board for Grid Biosciences.
Mr. Denny has authored and co-authored more than 200 peer-reviewed papers and serves on the editorial board of Communications in Cytometry and Journal of Clinical Virology. He holds an M.Sc in Molecular and Biomedical Immunology from the University of East London and a degree in Medical Law (M.Phil) from the Institute of Law and Ethics in Medicine, School of Law, University of Glasgow. In 1991, he completed a course of study in Strategic Management at The Wharton School, University of Pennsylvania. In 1993, he completed the Program for Advanced Training in Biomedical Research Management at Harvard School of Public Health. In December 2005, he was inducted as a Fellow into the College of Physicians of Philadelphia, the oldest medical society in the US.
While living in New Jersey, Mr. Denny was active in his community, gaining additional experience from two publicly elected positions. In 2000, Mr. Denny was selected by the New Jersey League of Municipalities to Chair the New Jersey Community Mental Health Citizens’ Advisory Board and Mental Health Planning Council as a gubernatorial appointment.
Chi Wei Cliburn Chan
Computational immunology (stochastic and spatial models and simulations, T cell signaling, immune regulation)
Statistical methodology for immunological laboratory techniques (flow cytometry, CFSE analysis, receptor-ligand binding and signaling kinetics)
Informatics of the immune system (reference and application ontologies, meta-programming, text mining and machine learning)
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