Detection of unseparated human lymphocytes by flow cytometry

The protocol for flow cytometry analysis presented here has been specifically developed for studies of human peripheral blood cells. In this protocol, analysis is performed on unseparated cells in whole peripheral blood, rather than on Ficoll-Hypaque-purified mononuclear cells. The advantage of this approach is that it requires less time, uses smaller blood volumes, and eliminates possible differential blood loss as a result of cell separation techniques. In this regard, B cell recovery using the whole blood method is significantly greater than that obtained using Ficoll-Hypaque-purified mononuclear cells. However, because lymphocytes generally represent a minority of peripheral cells (especially in adults), careful gating of the test samples for lymphocytes is a more critical requirement in this procedure than in other procedures using purified cells.