Determine the potency of BCG vaccines by flow cytometer

Abstract

The Bacille Calmette-Guerin (BCG) vaccine is a live attenuated vaccine prepared from a strain of Mycobacterium bovis. The potency of this vaccine is dependent on the amount of viable cells. The conventional method to determine the potency of the BCG vaccine is to measure the number of colony forming units (CFU) produced after the vaccine has been reconstituted in normal saline and the cells cultured on Lowenstein-Jensen (LJ) medium. However, as M. bovis is a slow growing organism, it needs to grow at least four weeks to be detected in the potency assay. The results of the potency assay could also vary due to manual counting. Therefore, in order to increase the assay efficiency and decrease the variation of the results, we developed an alternative method by using flow cytometer combined with cell counter (viable counts calculated from flow cytometer and counter, VFC) to determine the potency of BCG vaccines. To further test the accuracy of the assay we developed, the viable counts often batches of BCG vaccine determined by the VFC assay and conventional CFU method were compared. Wilcoxon statistical analysis showed that there were no statistically significant differences between the VFC assay and the conventional CFU method. Moreover, the results of the VFC assay are all read out from instruments, which could reduce the variation from different operators in order to overcome the limitation of the conventional CFU method. In summary, the results demonstrated that the potency of BCG vaccines could be determined by the VFC assay within four hours instead of four weeks cultivation by the conventional CFU method. On the whole, compared to the conventional CFU method, the VFC assay is a rapid, efficient and reliable method to determine the potency of BCG vaccines.