Evaluation of lymphocyte proliferation by immunohistochemistry in the local lymph node assay

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.