Evaluation of Methodologic Variables of the In Vitro Lymphocyte Transformation Assay

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Abstract
Optimum methodologic variables for assessing cellular immunity by in vitro lymphocyte transformation (LT) were determined using spherulin and coccidioidin antigens. This study was conducted in an area endemic for coccidioidomycosis and included healthy, coccidioidomycosis skin test positive (STP) and negative (STN) subjects, and patients with mild, acute disease. The authors examined the relationship between coccidioidin (1:100) and spherulin (low dose) skin test reactivity and lymphocyte transformation (LT) responses to the same antigens. Counts per minute (CPM) and stimulation index (SI) as methods of expressing tritiated thymidine uptake were compared. The LT assays were set up in duplicate test systems using autologous and homologous plasma. Both antigens differentiated between STP and STN groups (P ≤ 0.001–0.004), but values obtained with spherulin-induced LT were greater than those using coccidioidin (P < 0.001). Values in CPM and SI were greater in the spherulin-induced LT assay using autologous compared with AB plasma. Specifically, for detecting cellular immunity to coccidioidomycosis, the combination of spherulin-induced LT using autologous plasma and expressing the results in CPM gave the best discrimination between STP and STN subjects. Based on epidemiologic data, the latter method also appeared more sensitive than the skin test in detecting cellular immunity to coccidioidomycosis. In general, these data illustrate the variable effectiveness of different antigens for inducing LT responses and further show how different plasma sources affect the LT response. Finally, these data suggest that lymphocyte blast transformation results expressed as CPM may give more consistent values and better discrimination between immune and nonimmune subjects than results expressed as stimulation indices.