In vitro drug causality assessment in Stevens–Johnson syndrome – alternatives for lymphocyte transformation test
Werner J. Pichler, Department of Rheumatology, Clinical Immunology and Allergology, Inselspital, University Bern, CH-3010 Bern, Switzerland.
Patients with Stevens–Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) are often exposed simultaneously to a few potentially culprit drugs. However, both the standard lymphocyte transformation tests (LTT) with proliferation as the assay end-point as well as skin tests, if done, are often negative.
As provocation tests are considered too dangerous, there is an urgent need to identify the relevant drug in SJS/TEN and to improve sensitivity of tests able to identify the causative drug.
Fifteen patients with SJS/TEN with the ALDEN score ≥ 6 and 18 drug-exposed controls were included. Peripheral blood mononuclear cells (PBMC) were isolated and cultured under defined conditions with drugs. LTT was compared to the following end-points: cytokine levels in cell culture supernatant, number of granzyme B secreting cells by ELISpot and intracellular staining for granulysin and IFNγ in CD3+ CD4+, CD3+ CD8+ and NKp46+ cells. To further enhance sensitivity, the effect of IL-7/IL-15 pre-incubation of PBMC was evaluated.
Lymphocyte transformation tests was positive in only 4/15 patients (sensitivity 27%, CI: 8–55%). Similarly, with granzyme B-ELISpot culprit drugs were positive in 5/15 patients (sensitivity 33%, CI: 12–62%). The expression of granulysin was significantly induced in NKp46+ and CD3+ CD4+ cells (sensitivity 40%, CI: 16–68% and 53%, CI: 27–79% respectively). Cytokine production could be demonstrated in 38%, CI: 14–68% and 43%, CI: 18–71% of patients for IL-2 and IL-5, respectively, and in 55%, CI: 23–83% for IFNγ. Pre-incubation with IL-7/IL-15 enhanced drug-specific response only in a few patients. Specificities of tested assays were in the range of 95 (CI: 80–99%)–100% (CI: 90–100%).
Conclusions and Clinical Relevance
Granulysin expression in CD3+ CD4+, Granzyme B-ELISpot and IFNγ production considered together provided a sensitivity of 80% (CI: 52–96%) and specificity of 95% (80–99%). Thus, this study demonstrated that combining different assays may be a feasible approach to identify the causative drug of SJS/TEN reactions; however, confirmation on another group of patients is necessary.