Size distribution, electronic recognition, and counting of human blood monocytes

During a study on the separation of human blood monocytes from lymphocytes, a method was developed to recognize and count monocytes by electronic means. Lightscattering (Cytograf, Bio/Physics), and changes in electrical resistance (Channelyzer, Coulter) were used to size mononuclear leukocytes directly in cell suspensions. Both methods revealed a size distribution profile in which two populations of mononuclear leukocytes could be distinguished. The largest cells were virtually eliminated after phagocytosis of iron particles. We confirmed that these cells were monocytes by three different criteria: the intracellular lysozyme activity, the number of phagocytes, and the percentage of cells with kidney-shaped nuclei. The highly significant correlations we found showed that monocytes could be recognized and counted by electronic sizing. For this method, purified mononuclear leukocyte preparations had to be used, since the presence of erythrocytes, platelets, and polymorphonuclear cells interfered. Statistical analysis revealed that electronic sizing permitted discrimination of differences in monocyte content of 4.5%, with a probability of 95%. It was calculated that this sensitivity of electronic monocyte counting was about three times higher than the sensitivity of microscopic methods. Since 100,000 cells can be sized within a few seconds, not only the efficiency of the preparation but also minor changes in the size of monocytes and lymphocytes introduced during the isolation can be followed.