REDUCTION OF ESCAPEE FORMATION IN FLOW CYTOMETRIC ANALYSIS OF LYMPHOCYTE SUBSETS

Most experienced flow cytometrists performing immunophenotyping of lym phocyte subsets are aware of the escapee phenomenon, in which some pos itive cells are found outside a lymphocyte gate based on forward and r ight angle scatter. However, little information is available on the le vels of escapees formed with different antibodies, the roles of fluoro chromes and lysing agents, the mechanism explaining the phenomenon, or methods to reduce it. We thus performed a systematic analysis of the escapee phenomenon to clarify these issues. A panel of monoclonal anti bodies, including a phycoerythrin (PE) conjugate and a fluorescein iso thiocyanate (FITC) conjugate of the same antibody from one manufacture r, was used to treat whole blood specimens, after which red cells were lysed using 0.15 M ammonium chloride (AmChl). The percentages of gate d lymphocytes expressing CD3, CD8, CD19, and HLA-DR, but not CD2, CD4, CD16, and CD25, were significantly lower in FITC-stained versus PE-st ained preparations. Correlated analysis of green fluorescence and forw ard scatter showed that, on average, 18% of CD3(+) events, 24% of CD8( +) events, and 25% of CD19(+) events were escapees when using the FITC conjugate. In dual color analysis, CD3(+) escapees were positive for CD62-P, CD13, and CD14, indicating that the escapee events consisted o f FITC-anti-CDS-coated lymphocytes complexed with platelet-coated myel oid cells. In studies of the role of lysing agent, essentially no esca pees were found in specimens treated with FAGS lysing solution, which contains formaldehyde. We therefore included a similar denaturing agen t, paraformaldehyde (0.1%), in the AmChl lysing agent, and found that the occurrence of escapees was markedly reduced. These findings show t hat the escapee phenomenon occurs when using some FITC-conjugated mono clonal antibodies in conjunction with AmChl lysing agent, and can be r educed by inclusion of paraformaldehyde in the lysing agent.