Design and analysis considerations in evaluating the chemiluminescence response of mouse spleen cells

Chemiluminescence is the result of the respiratory burst generated by phagocytic cells after stimulation by antigen. The measurement of chemiluminescence represents a sensitive means for detecting antigenic stimulation and immune cell function. Although the kinetics of chemiluminescence reactions have been described, appropriate statistical methods for the evaluation of data from chemiluminescence assays have not been reported. Based on examination of data from several studies in which the chemiluminescence response of spleen cells was investigated after stimulation with the particulate antigen zymosan, recommendations are made for the design and statistical evaluation of such studies. Three parameters were used in assessing the chemiluminescence response; peak intensity of the emitted light, time to peak, and the area under the intensity-time curve. The data indicated that peak intensity alone provides an adequate characterization of the chemiluminescence response. Since percent change in response upon treatment is of interest, analysis on the log scale is appropriate, and the statistical procedure of choice in evaluating data of this type is a trend analysis. The need for a balanced allocation of treatments to avoid potential bias is demonstrated. The methods proposed are illustrated with data from two studies in which the effect of preincubation with low concentrations of ketoconazole, an antifungal agent, on the chemiluminescence response of BALB/cBY spleen cells was examined.