Development and initial validation of a modified LTT assay in patients with DRESS and AGEP
Research Square (Research Square)(2022)
摘要
Abstract BackgroundThe lymphocyte transformation test (LTT) is an in vitro assay used to diagnose drug induced hypersensitivity reactions by detecting the activation and expansion of drug-specific memory T cells to the suspected implicated drug. Traditionally radiolabelled thymidine (3H-thymidine) has been used but requires the handling and disposal of radioactive materials.ObjectiveTo examine safe alternatives to 3H-thymidine, test assay modifications for improved assay sensitivity and validate the modified LTT in patients with DRESS and AGEP.MethodsFour proliferation detection assays (BRDU, CyQUANT™, MTT and XTT) were screened for LTT sensitivity. XTT the most sensitive and practical was selected for further validation. Modifications like autologous serum (AS) and regulatory T cell depletion (T-REG) were evaluated for improved assay sensitivity. Finally, an initial validation of the XTT-LTT was performed in 8 patients with DRESS and 2 with AGEP including cytokine testing.ResultsOf the non-radioactive alternatives we tested, XTT a colorimetric assay was the most sensitive and practical to move to validation. The addition of AS increased background signal. Depletion of T-REGs improved sensitivity but cell sorting time and risk of contamination limited benefit.Of eight patients diagnosed with DRESS and 2 with AGEP tested with XTT-LTT assay results showed our assay matched clinical findings of implicated drugs in 8/10 patients when using a stimulation index (SI) ≥2 and 8/10 with analysis by ANOVA. All ten patients were correctly diagnosed by either analysisConclusionXTT appears to be a safe, viable alternative to 3H-thymidine, with high sensitivity and allowing direct cytokine quantification on specific patient cells.
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关键词
ltt assay,dress
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