Use of CD45 fluorescence and side-scatter characteristics for gating lymphocytes when using the whole blood lysis procedure and flow cytometry.

The light-scatter characteristics of lymphocytes are commonly used to gate lymphocytes for further analysis in a lysed whole-blood assay. Because lymphocytes can be identified by antigens that they possess, a light-scatter gate can be validated by measuring parameters other than light scatter. When a specimen possesses poor light scatter (usually from contaminating nonlymphocytes within the light-scatter gate for lymphocytes), the quality of the gate and, thus, the analyses of lymphocyte subsets can be compromised. We present data to demonstrate the use of CD45 fluorescence combined with side scatter (SSC) for analyzing lysed whole-blood specimens. When we compared CD45/SSC to light scatter (forward and side scatter) for validating a lymphocyte gate, both methods performed similarly in recovering as many lymphocytes as possible in the gate (lymphocyte recovery); however, the CD45/SSC gate had fewer contaminants within the gate (lymphocyte purity). Replicate CD3 values from the CD45/SSC gate were less variable than those from the light-scatter gate, confirming that most of the variability in a light-scatter gate is due to nonlymphocyte contaminants in the gate. We propose that lymphocytes be gated using CD45 fluorescence as well as side-scattering properties and that CD3 also be included in each data analysis tube for quality control.