Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry

It is possible accurately to distinguish lymphocytes from other leukocyte populations in peripheral blood using the combination of fluorescence associated with CD45/CD14 and forward and orthogonal light scatter. By identifying the cell population of interest based on immunofluorescence, a light scattering window can then be drawn to include all (greater than or equal to 98%) of the lymphocytes. In this manner, maximal recovery of the lymphocytes within a sample can be consistently obtained. The combination of light scattering and immunofluorescence can also be used to define the purity of the gate. The identification of nonlymphocytes within the light scattering gate can then be used to establish an accurate denominator for the percent lymphocytes stained. Once the optimal data acquisition gate has been established and characterized, it is possible to correct subsequent analyses with that particular sample since the reactivity of monoclonal antibodies on monocytes and granulocytes can be accounted for once the nonlymphocytes have been identified as being within the acquisition gate.