(220b) Immunophenotyping of Leukocytes on Antibody Microarrays

Leukocytes are a heterogeneous mixture of several cell subsets that play an important role in fighting against injuries, infections, malignancies and autoimmune disorders. Flow cytometry is the standard technology for the analysis of leukocyte subsets; however, this technology is expensive; it is not suitable for analysis of small cell populations; it provides only limited cell morphology data, and it is not designed for time-based studies of cells of interest. This presentation describes design of a solid-phase cytometry platform based on printed microarrays of leukocyte specific monoclonal antibodies (mAbs). Printing antibodies in an array format allowed capturing specific leukocyte subsets in pre-defined locations on the substrate for further characterization and enumeration. Based on their importance in HIV detection and monitoring, the microarray was designed to capture primary human CD4+ and CD8+ T-cells from peripheral blood. The microarrays comprised of 150 µm diameter spots of anti-CD4, -CD8, anti-mouse IgG (negative control) Abs, and poly-lysine (positive control) were printed using a robotic arrayer on glass slides pre-coated with poly (ethylene glycol) hydrogel (PEG). PEG coating proved to be effective in eliminating non-specific leukocyte adhesion to the glass surface. Red blood cell (RBC) depleted whole blood was incubated with antibody microarrays for 15 minutes and was then placed in a miniature flow chamber for controlled washing. Flow rates and shear stress conditions required to remove non-specifically (loosely) bound leukocytes were determined. The captured cells were identified by immunofluorescent staining as CD4+ and CD8+ lymphocytes with a purity exceeding 95%. Lysine spots captured leukocytes indiscriminately and the surface cell density on lysine corresponded well to the overall leukocyte concentration provided by hemocytometer. Therefore, ligand microarrays captured pure CD4 and CD8 T cells, yielded overall cell concentration in the sample and provided negative (isotype) controls. The Ab microarray-based cytometry platform described here offers a quick, simple and inexpensive means for capturing and enumerating leukocyte subsets with minimal sample preparation and from small cellular samples. This solid-phase cytometry method is currently being applied for monitoring CD4 and CD8 T cell counts in HIV infections.