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Development and initial validation of a modified lymphocyte transformation test (LTT) assay in patients with DRESS
Chris Weir1,2, Jamma Li3,4, Richard Fulton4
1Northern Blood Research Laboratory, Kolling Institute of Medical Research, Royal North Shore Hospital and the Sydney Medical School, University of Sydney, Sydney, NSW, Australia. chris.weir@sydney.edu.au.
Background:
The 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.
Objective:
To examine safe alternatives to 3H-thymidine, test assay modifications for improved assay sensitivity and evaluate the modified LTT in patients with DRESS and AGEP.
Methods:
Four proliferation detection assays (BRDU, CyQUANT™, MTT and XTT) were screened for LTT sensitivity. XTT the most sensitive and practical was selected for further evaluation Modifications like autologous serum (AS) and regulatory T cell depletion (T-REG) were tested for improved assay sensitivity. Finally, an initial evaluation of the XTT-LTT was performed in 8 patients with DRESS and 2 with AGEP including cytokine testing.
Results:
Of the non-radioactive alternatives we tested, XTT a colorimetric assay was the most sensitive and practical to move to evaluation. 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 analysis.
Conclusion:
XTT 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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