Predictive capacity of a non-radioisotopic local lymph node assay using flow cytometry, LLNA: BrdU-FCM: Comparison of a cutoff approach and inferential statistics

Da Eun Kim, Hyeri Yang, Won Hee Jang, Kyoung Mi Jung, Miyoung Park, Jin Kyu Choi, Mi Sook Jung, Eun Young Jeon, Yong Heo, Kyung Wook Yeo, Ji Hoon Jo, Jung Eun Park, Soo Jung Sohn, Tae Sung Kim, Il Young Ahn, Tae Cheon Jeong, Kyung Min Lim, Seung Jin Bae

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


In order for a novel test method to be applied for regulatory purposes, its reliability and relevance, i.e., reproducibility and predictive capacity, must be demonstrated. Here, we examine the predictive capacity of a novel non-radioisotopic local lymph node assay, LLNA:BrdU-FCM (5-bromo-2'-deoxyuridine-flow cytometry), with a cutoff approach and inferential statistics as a prediction model. 22 reference substances in OECD TG429 were tested with a concurrent positive control, hexylcinnamaldehyde 25%(PC), and the stimulation index (SI) representing the fold increase in lymph node cells over the vehicle control was obtained. The optimal cutoff SI (2.7 ≤ cutoff < 3.5), with respect to predictive capacity, was obtained by a receiver operating characteristic curve, which produced 90.9% accuracy for the 22 substances. To address the inter-test variability in responsiveness, SI values standardized with PC were employed to obtain the optimal percentage cutoff (42.6 ≤ cutoff < 57.3% of PC), which produced 86.4% accuracy. A test substance may be diagnosed as a sensitizer if a statistically significant increase in SI is elicited. The parametric one-sided t-test and non-parametric Wilcoxon rank-sum test produced 77.3% accuracy. Similarly, a test substance could be defined as a sensitizer if the SI means of the vehicle control, and of the low, middle, and high concentrations were statistically significantly different, which was tested using ANOVA or Kruskal-Wallis, with post hoc analysis, Dunnett, or DSCF (Dwass-Steel-Critchlow-Fligner), respectively, depending on the equal variance test, producing 81.8% accuracy. The absolute SI-based cutoff approach produced the best predictive capacity, however the discordant decisions between prediction models need to be examined further.

Original languageEnglish
Pages (from-to)76-84
Number of pages9
JournalJournal of Pharmacological and Toxicological Methods
StatePublished - 1 Mar 2016

Bibliographical note

Funding Information:
This research was supported by a grant ( 13172MFDS987 ) from the Ministry of Food and Drug Safety of Korea.

Publisher Copyright:
© 2015 Elsevier Inc.


  • Descriptive and inferential statistics
  • Local lymph node assay
  • Prediction model
  • Predictive capacity
  • Skin sensitization


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