METAL ALLERGY TEST
METAL ALLERGY TEST
What is an LTT Assay?
How is the metal-LTT test conducted?
Why use metal-LTT?
This assay facilitates a dose response quantification of metal-induced hypersensitivity responses in terms of generalized lymphocyte reactivity (i.e. proliferation). Issues of sensitivity and specificity remain unresolved as well as how implant performance is related to positive reactivity results. Metal-specific reactivity is gauged by comparing non-treated to treated lymphocytes from the same individual and categorized using the following general criteria: 2-4 fold response =mild reactivity, 5-8 fold =moderate reactivity, and >8 =high reactivity.
Technical Protocol: Proliferation Assay (Lymphocyte Transformation Tests): Proliferation of cells is measured by [3H]-thymidine (Amersham International, Arlington Heights, IL) incorporation into DNA in a 96-well microplate system. The average for each treatment is normalized to that of the negative control (no treatment) producing a ratio, generally termed a proliferation factor, proliferation index, proliferation ratio or stimulation index, SI. The SI is used to compare lymphocyte reactivity to the different metals. The lower limit of this stimulation index is zero indicating all cells stopped dividing before addition of [3H]-thymidine, after 5 days. Proliferation assays are performed using Ficoll separated peripheral blood mononuclear cells (PBMCs) collected from 30-40 milliliters of peripheral blood. These isolated PBMCs are cultured in 96-well cell-culture plates (Sigma), at a density of 0.1-0.3x10^6 cells/well for a period of 6 days in 150
L of DMEM/well, 10% autologous serum at 37degrees C and 0.5% CO2, with metal treatments, a positive control (0.01 mg/ml PHA) and a negative control (untreated). Each treatment is conducted in triplicate (3 wells/treatment). [3H]-thymidine is added during the last 12 hours of incubation after 5 days of treatment. At day six [3H]-thymidine uptake (1
Ci/culture well) is measured using liquid scintillation. The SI is calculated using measured radiation counts per minute (cpm): Simulation Index = (mean cpm with treatment) / (mean cpm without treatment).
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