Setting objective thresholds for rare event detection in flow cytometry.
J Immunol Methods
; 409: 54-61, 2014 Jul.
Article
em En
| MEDLINE
| ID: mdl-24727143
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 Fß 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 Fß method performs consistently across different centers, samples, and instruments because it optimizes the precision/recall tradeoff by using both negative and positive controls.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Leucócitos Mononucleares
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Monitorização Imunológica
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Citocinas
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Ensaio de Proficiência Laboratorial
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Citometria de Fluxo
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Laboratórios
Tipo de estudo:
Clinical_trials
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Diagnostic_studies
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Guideline
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Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article