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Abstract: The antibody (Ab) array format provides a unique opportunity to pan and characterize multiple leukocyte subsets in parallel. However, the questions of reproducibility and robustness of leukocyte panning on Ab arrays need to be answered for this technology to become an immunophenotyping tool. The present study sought to address several of these questions, including: (1) purity of leukocyte subsets captured on Ab regions, (2) dynamics of leukocyte binding, (3) elimination of non-specific cell adhesion, and (4) standardization of cell washing conditions. Abs for CD4 T-cells, CD8 T-cells, CD36 monocytes, and CD16b neutrophils were dispensed onto standard glass slides containing a thin film of poly(ethylene glycol) (PEG) hydrogel. PEG gel coating was highly effective in eliminated non-specific cell adhesion on the surface. Incubation of the Ab arrays with red blood cell (RBC) depleted whole blood resulting in antigen-specific panning of leukocyte subsets on the respective Ab domains. A flow through chamber was employed to determine optimal shear stress conditions for removal of non-specifically attached cells. The purity of the four subsets remaining on the surface after washing was determined by Wright staining and immunofluorescence, and was found to be as follows: CD4 T-cells (99.2±0.3%), CD8 T-cells (98.7±0.3%), CD36 monocytes (97.2±0.9%), and CD16b neutrophils (99.1±0.6%). In conclusion, the methods described in this study allow to separate whole blood into pure leukocyte subsets with minimal sample preparation and handling. These approaches will be valuable in the future development of Ab arrays as tools for quantitative immunophenotyping of leukocytes. [Copyright &y& Elsevier]