Method:
(1) Lymphocytes are separated from whole blood using a Ficoll-Hypaque gradient.
(2) The lymphocytes are placed in a medium containing labelled thymidine. In the past this was often carbon-14. Newer techniques used labels that can be measured by immune-based assays
(3) The material in question is then added to an aliquot of the lymphocyte solution and the sample is incubated.
(4) A control (usually a similar sample of lymphocytes exposed to a standard material that is nonstimulating) is run in parallel.
(5) Following incubation the lymphocytes are collected onto glass fiber filters.
(5) The uptake of thymidine incorporated into the lymphocyte DNA is then measured. For radioactive labelled thymidine, this is done in a scinitillation counter, with results expressed as counts per minute per 100,000 lymphocytes. For immune-based assays flow cytometry may be used.
stimulation index =
= (measurement for target material) / (measurement for control material)
Interpretation:
• A stimulation index = 1 indicates that the material is nonstimulatory.
• A stimulation index < 1 indicates that the test material is lymphocytoxic.
• The level of the SI to indicate a "positive" test varies with the test material. For lymphocytes exposed to varicella-zoster viral antigen, an SI > 3 indicates that the subject had a previous exposure to the virus. For exposure to a mitogen the SI needed to be considered a positive reaction is > 50.
• A result > 1 but less than the "positive" level can be considered borderline.
Limitations:
• Patients with HIV infection may show significant variability in the counts for unstimulated lymphocytes. In such cases testing can be done by comparing patient results to that of a normal donor. A drawback is that sequential testing may require access to the same normal donor over a period of time.
