Appraisal of within- and between-laboratory reproducibility of non-radioisotopic local lymph node assay using flow cytometry, LLNA:BrdU-FCM: comparison of OECD TG429 performance standard and statistical evaluation.
Mouse local lymph node assay (LLNA, OECD TG429) is an alternative test replacing conventional guinea pig tests (OECD TG406) for the skin sensitization test but the use of a radioisotopic agent, (3)H-thymidine, deters its active dissemination. New non-radioisotopic LLNA, LLNA:BrdU-FCM employs a non-radioisotopic analog, 5-bromo-2'-deoxyuridine (BrdU) and flow cytometry. For an analogous method, OECD TG429 performance standard (PS) advises that two reference compounds be tested repeatedly and ECt(threshold) values obtained must fall within acceptable ranges to prove within- and between-laboratory reproducibility. However, this criteria is somewhat arbitrary and sample size of ECt is less than 5, raising concerns about insufficient reliability. Here, we explored various statistical methods to evaluate the reproducibility of LLNA:BrdU-FCM with stimulation index (SI), the raw data for ECt calculation, produced from 3 laboratories. Descriptive statistics along with graphical representation of SI was presented. For inferential statistics, parametric and non-parametric methods were applied to test the reproducibility of SI of a concurrent positive control and the robustness of results were investigated. Descriptive statistics and graphical representation of SI alone could illustrate the within- and between-laboratory reproducibility. Inferential statistics employing parametric and nonparametric methods drew similar conclusion. While all labs passed within- and between-laboratory reproducibility criteria given by OECD TG429 PS based on ECt values, statistical evaluation based on SI values showed that only two labs succeeded in achieving within-laboratory reproducibility. For those two labs that satisfied the within-lab reproducibility, between-laboratory reproducibility could be also attained based on inferential as well as descriptive statistics.
College of Pharmacy, Ewha Womans University, Seoul 120-808, Republic of Korea.
Hyeri
H
Yang
College of Pharmacy, Ewha Womans University, Seoul 120-808, Republic of Korea.
Jihye
J
Na
Medical Beauty Research Division, Amorepacific Corp. R&D Center, Republic of Korea.
Won-Hee
WH
Jang
Pharmacology Efficacy Team, Biotoxtech Co., Ltd., Republic of Korea.
Mi-Sook
MS
Jung
Pharmacology Efficacy Team, Biotoxtech Co., Ltd., Republic of Korea.
Jun-Young
JY
Jeon
Department of Occupational Health, College of Natural Sciences, Catholic University of Daegu, Daegu, 712-702, Republic of Korea.
Yong
Y
Heo
Department of Occupational Health, College of Natural Sciences, Catholic University of Daegu, Daegu, 712-702, Republic of Korea.
Kyung-Wook
KW
Yeo
Department of Occupational Health, College of Natural Sciences, Catholic University of Daegu, Daegu, 712-702, Republic of Korea.
Ji-Hoon
JH
Jo
College of Pharmacy, Ewha Womans University, Seoul 120-808, Republic of Korea. Electronic address: kmlim@ewha.ac.kr.
Kyung-Min
KM
Lim
College of Pharmacy, Ewha Womans University, Seoul 120-808, Republic of Korea. Electronic address: sjbae@ewha.ac.kr.
SeungJin
S
Bae
D000818:Animals; D001973:Bromodeoxyuridine; D005434:Flow Cytometry / Q000379:methods / Q000592:standards; D023441:Local Lymph Node Assay; D051379:Mice; D012015:Reference Standards; D015203:Reproducibility of Results
D001973:Bromodeoxyuridine
Descriptive and inferential statistics; LLNA:BrdU-FCM; Local lymph node assay; OECD TG performance standard; Within and between laboratory reproducibility