Shortcomings in the application of multicolour flow cytometry in lymphocyte subsets enumeration

The lymphoid system is composed of numerous phenotypically distinct subsets of cells, each of which has a unique role in the effectiveness of an immune response. To distinguish specifically between these subsets, it is mandatory to detect simultaneously different cell surface antigens. This became feasible by the development of multicolour flow cytometric technologies. With these techniques, researchers now have the opportunity to study individual cells in far greater detail than previously possible. However, proper data analysis, interpretation and presentation of results will require a high level of understanding of the intricacies of the technology and the inherent limitations of the acquired data. The present report is intended to contribute to the better understanding of how the flow cytometer operates. This report may help new and inexperienced users to work appropriately with the flow cytometer.