Light scatter based lymphocyte gate–helpful tool or source of error?

During flow cytometric analysis of 29 samples of human peripheral blood mononuclear cells, we observed that a light scatter-based lymphocyte gate would fail to cover all TCR (CD3)-expressing cells. A mean of 7.9 +/- 5.3% of all CD3+ cells was found outside the gate. In addition, this shifted cell subset showed an altered CD4/CD8 ratio (8.1 +/- 5.0% of all CD4+ and 13.7 +/- 5.8% of all CD8+ cells). The use of standard light scatter-based lymphocyte gates in acquisition and/or analysis of flow cytometry (FCM) data may thus lead to erroneous results, particularly if T cells are to be studied.