Reproducibility of leucocyte migration from agarose microdroplets.

The 18-hour migration of leucocytes from 31 healthy persons was measured in 33 experiments by the agarose microdroplet method using 15-26 replicate droplets in separate culture wells in each experiment. A comparison of the measurement of the migration of leucocytes by a formula derived from two diameters of the droplets and by planimetry of the projected image of droplets, demonstrated the same wide range of coefficients of variation for the two methods and therefore no important difference in their accuracy. In using a ratio, the migration index, to express the size of differences between experimental groups of cultures, the correct choice of cut-off value demands knowledge of the distribution of that ratio amongst cultures no inhibited by the inclusion of "antigen" or other agent. This could not be ascertained from the observed data because of the practical limitation on the number of samples obtainable in each test. A computer simulation of the sampling process was used to determine this distribution and to evaluate statistical cut-off points. The size of the sample required for conventional degrees of accuracy is shown to vary according to the size of the mean migration area and its standard deviation in unstimulated control cultures. In the study 25/33 (76%) experiments would have been adequately assessed if 30 replicates had been used in every case; if 5 replicates had been used only 2/33 (6%) experiments would have been controlled. In the literature upon leucocyte migration, only 3 or 4 replicates of the control and test cultures are generally described and a cut-off migration index of 0.8 is usually accepted arbitrarily. Our observations indicate that this is an inefficient way of assessing data in such a variable system.