Setting objective thresholds for rare event detection in flow cytometry
Abstract
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. © 2014 Elsevier B.V.
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https://hdl.handle.net/10161/14706Published Version (Please cite this version)
10.1016/j.jim.2014.04.002Publication Info
Richards, A; Staats, Janet; Enzor, Jennifer; McKinnon, K; Frelinger, J; Denny, Thomas
Norton; ... Chan, C (2014). Setting objective thresholds for rare event detection in flow cytometry. Journal of Immunological Methods, 409. pp. 54-61. 10.1016/j.jim.2014.04.002. Retrieved from https://hdl.handle.net/10161/14706.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
Thomas Norton Denny
Professor in Medicine
Thomas N. Denny, MSc, M.Phil, is the Chief Operating Officer of the Duke Human Vaccine
Institute (DHVI) and the Center for HIV/AIDS Vaccine Immunology (CHAVI), 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. He has recently been
appointed to the Duke University Fuqua School of Business Health Sector Advisory Council.
Previously, he was an Associate Professor of Pathology, Laboratory M
Kent James Weinhold
Joseph W. and Dorothy W. Beard Distinguished Professor of Experimental Surgery
In addition to their ongoing HIV/AIDS-related research activities, the Weinhold Laboratory
is focused on utilizing a comprehensive repertoire of highly standardized and formerly
validated assay platforms to profile the human immune system in order to identify
immunologic signatures that predict disease outcomes. These ongoing studies span a
broad range of highly relevant clinical arenas, including: 1) cancer (non-small cell
lung cancer, head and neck cancer, glioblastoma neof
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