Evaluation of a method for counting absolute numbers of cells with a flow cytometer

We evaluated a method for performing absolute cell counts of lymphocyte populations with a flow cytometer. In this method, TruCount, test tubes that contain a known number of brightly fluorescent polystyrene beads are provided by the manufacturer. Whole anticoagulated blood is accurately pipetted into the tubes and mixed with fluorochrome-labeled monoclonal antibodies, the erythrocytes are lysed, and this mixture is analyzed on the flow cytometer. Absolute counts of lymphocyte subsets are calculated by determining the ratio of beads to the cell population of interest and then multiplying this ratio by the number of beads in the tube. We found this method to be reproducible. The values we obtained by the TruCount method were 5 to 10% higher than those obtained by conventional methods (flow cytometry and automated hematology) used to determine absolute numbers of cells. We believe that these differences are due to the methods of determining absolute cell counts and not to faulty identification of lymphocyte subsets.