Statistical criteria to establish optimal antibody dilution in flow cytometry analysis.

BACKGROUND In direct techniques of flow cytometry, the optimal antibody dilution or titer point is established from the plateau area of the antibody titration curve. However, the plateau area is defined without any statistical criteria, which may lead to an incorrect selection of antibody dilution. Herein, we report statistical criteria to establish the optimal antibody dilution for CD14, CD8, CD4, and CD3 analysis by flow cytometry in peripheral whole blood. METHODS The unpaired t-test (two-tail P value) was used as statistical criteria to analyze the titration curve of the following monoclonal antibody panels: CD14-FITC, CD8-FITC, CD4-RD1, and CD3-PC5. RESULTS Using the unpaired t-test (two-tail P value), the plateau area from the antibody titration curve was fitted when two consecutive antibody volumes showed mean peak of channel fluorescence (MPCF) values not significantly different. When the antibody was used at volume corresponding to that of the antibody titration point, the flow cytometry analysis of whole blood samples with different density of cell antigens can be correctly discriminated. CONCLUSION This statistical criteria allows the fitting of the plateau area of MPCF versus antibody volume and consequently, to define the optimal antibody dilution.