Objective  To establish a 14-color flow cytometry protocol for the examination of leukocyte subsets in human peripheral blood.

  Methods  We used cell membrane surface antibodies CD45, CD3, CD4, CD8, CD19, CD56, CD16, CD14, CD25, CD127, HLA-DR, CD123, CD11c and nucleus staining dye DAPI to establish a 14-color flow cytometry assay to determine the major cell subsets in human peripheral blood. We collected peripheral blood specimens from healthy volunteers to test for antibody titers and optimal photomultiplier tube (PMT) voltage, and to conduct single-color staining and fluorescence minus one control staining. After determining the test method and test conditions, the peripheral blood samples of 18 healthy volunteers were analyzed.

  Results  According to the cell classification and staining index, optimal antibody mass concentrations selected were as follows: CD25 and CD127 at 8.0 μg/mL, CD45, CD3, CD14 and CD123 at 4.0 μg/mL, CD8, CD19, CD56, CD16, HLA-DR and CD11c at 2.0 μg/mL, CD4 at 1.0 μg/mL and DAPI at 0.1 μg/mL. The detection voltages for CD45, CD3, CD4, CD8, CD19, CD56, CD16, CD14, CD25, CD127, HLA-DR, CD123, CD11c and DAPI were 450 V, 410 V, 400 V, 550 V, 405 V, 500 V, 520 V, 550 V, 550 V, 400 V, 450 V, 400 V, 580 V, and 300 V, respectively. The appropriate fluorescence compensation was determined by single-color staining and fluorescence minus one controls. The 14-color flow cytometry panel was established to analyze the main subsets of leukocytes in human peripheral blood, and peripheral blood samples from 18 healthy adults were examined, obtaining the percentages of each subset of peripheral blood leukocytes and the immunophenotypes of the main subsets.

  Conclusion  We established a 14-color panel for determining leukocyte subsets in human peripheral blood by flow cytometry, which produced stable and reliable results and was easy to operate.