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Updated: Aug 8, 2026
Isolation and Activation of Murine Lymphocytes
Published on: October 30, 2016
Evaluation of lymphocyte proliferation by immunohistochemistry in the local lymph node assay
C Boussiquet-Leroux1, G Durand-Cavagna, K Herlin
1Laboratoires Merck Sharp & Dohme-Chibret, Department of Safety Assessment, Research Center, Riom, France.
Insights
A modified local lymph node assay (LLNA) uses bromodeoxyuridine for lymphocyte proliferation detection. This enhanced method improves specificity and reproducibility for assessing chemical sensitizers in safety evaluations.
Area of Science:
- Toxicology
- Immunology
- Dermatology
Background:
- The local lymph node assay (LLNA) is a standard method for assessing chemical sensitization.
- Modifications are needed to improve its sensitivity, specificity, and routine applicability.
Purpose of the Study:
- To present a modified LLNA using bromodeoxyuridine (BrdU) labeling for enhanced lymphocyte proliferation detection.
- To evaluate the efficacy of this modified LLNA in identifying skin sensitizers and irritants.
Main Methods:
- A modified LLNA incorporating bromodeoxyuridine (BrdU) labeling of proliferating lymphocytes.
- Cell counting performed on mid-sagittal sections of lymph nodes via light microscopy.
- Testing of irritants, allergens, and a chemical of unknown potential using short and long protocols.
Main Results:
- The modified LLNA detected T cell proliferation with irritants in the short protocol, but not the long.
- Potassium dichromate showed a strong response in the short protocol.
- Weak to moderate sensitizers were better identified using the long protocol, while nickel sulfate was negative.
- Histological examination confirmed the absence of intercurrent processes.
Conclusions:
- The modified LLNA, using BrdU and restricted cell counting, offers increased specificity and sensitivity.
- The method is suitable for routine safety assessment, providing reproducible and statistically analyzable results.
- This non-isotopic approach serves as a valuable adjunct to the traditional LLNA.
Abstract:
A modified version of the local lymph node assay (LLNA) is presented, using bromodeoxyuridine to label proliferating lymphocytes. Cell counting is done on mid-sagittal sections of individual nodes under light microscopy. Two irritants (sodium lauryl sulfate and salicylic acid), four allergens of various sensitizing potential (potassium dichromate, 4-chloroaniline, neomycin sulfate and nickel sulfate) and one chemical of unknown sensitizing potential (ethyl 3-aminobenzoate) were tested either in the short protocol using three daily topical applications and/or in the long protocol with a pre-exposure step under an occluded patch. A weak T cell proliferation was noted with both irritants in the short protocol, but not in the long protocol. Potassium dichromate induced a strong proliferative response in the short protocol. A lesser sensitizing potential was detected for 4-chloroaniline, ethyl 3-aminobenzoate and neomycin sulfate but only in the long protocol. Nickel sulfate was negative in both protocols. The long protocol was the most valuable for weak or moderate sensitizers. Histological examination of nodes ruled out intercurrent processes. The present procedure offers several advantages. The use of a non-isotopic marker enables this test to be run in a routine safety assessment department and allows the preparation of permanent slides. An increased specificity is obtained by restricting cell counting to the paracortex. Moreover, the collection of individual data permits statistical analysis of the results. This method is sensitive and reproducible and may be viewed as a useful adjunct to the LLNA.
