Reproducibility, efficacy and methodology of mitogen-induced lymphocyte transformations by the whole blood assay

For the past few years, the whole blood culture method has been scrutinized for its usefulness and reproducibility in evaluating the immunologic status of patients. This method, used in our laboratory mainly screening specimens for evidence of immunodeficiencies, has been systematically evaluated for reproducibility, and factors affecting normal response through testing specimens from over 70 normal persons. Three mitogens (phytohemagglutinin, Concanavalin A, and pokeweed) were studied; peak time responses occurred later than in the separated cell culture method, and they were different for each mitogen. The dose response curves also depended on the mitogen tested, with pokeweed giving a sharp curve and the others a broad plateau. The harvesting procedure was studied by varying reagents and was optimized for speed and efficiency. The mononuclear cell count of the specimen had little effect on the results as long as it remained less than 3.5 X 10(6)/ml. The results for all mitogens followed a similar distribution curve irrespective of whether the results were expressed as cpm or cpm divided by the mononuclear cell count. The use of the stimulation ratio method to express results was less satisfactory. The mean coefficient of variation for all triplicate samples remained 20% or less and for all conditions tested. In evaluating the immunodeficient patient, the whole blood culture method was found to be equally as informative and easier to perform than the separated cell method.