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Please cite this paper as: Para-phenylenediamine-specific lymphocyte activation test: a sensitive in vitro assay to detect para-phenylenediamine sensitization in patients with severe allergic reactions. Experimental Dermatology 2010; 19: 435–441. Patients sensitized to para-phenylenediamine (PPD) by semi-permanent tattoos increasingly develop threatening allergic reactions in response to black hair dye. The gold standard to diagnose allergic contact dermatitis is to perform epicutaneous patch tests, however, iatrogenic sensitizations and severe patch test reactions to PPD have been described, the latter especially in patients with severe allergic reactions. We examined nine patients with severe allergic reactions in response to permanent hair dyes. Patch tests using the standard concentration of 1% or 0.5% PPD resulted in severe and sometimes even bullous reactions in all patients responsive to PPD. Titration revealed that at 1% of the standard concentration (0.01% PPD), patch test sensitivity decreased and only 50% of patients responded. Consequently, we established an in vitro assay to diagnose PPD allergy. Freshly isolated peripheral blood mononuclear cells (PBMC) were cultured with titrated concentrations of PPD with or without IL-2 supplementation, and cell proliferation was determined by [3H]-thymidine incorporation. Lymphocyte activation test (LAT ) detected PBMC cell proliferation specific to PPD, with at least 3.5-fold increase in [3H]-thymidine uptake in all PPD allergic patients. Most importantly, PPD –LAT without IL-2 supplementation remained negative in three out of eight PPD allergic patients. Thus, PPD-LAT with IL-2 supplementation demonstrated a sensitivity of 100%, remained unresponsive in controls not sensitized to PPD, and in one patient sensitive to other p-amino compounds. These data demonstrate that LAT with PPD can be used to detect PPD sensitization as a possible alternative to patch testing at least in patients with severe allergic reactions to PPD. [ABSTRACT FROM AUTHOR]