Reduction of escapee formation in flow cytometric analysis of lymphocyte subsets

Most experienced flow cytometrists performing immunophenotyping of lymphocyte subsets are aware of the escape phenomenon, in which some positive cells are found outside a lymphocyte gate based on forward and right angle scatter. However, little information is available on the levels of escapees formed with different antibodies, the roles of fluorochromes 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 antibodies, including a phycoerythrin (PE) conjugate and a fluorescein isothiocyanate (FITC) conjugate of the same antibody from one manufacturer, was used to treat whole blood specimens, after which red cells were lysed using 0.15 M ammonium chloride (AmChl). The percentages of gated lymphocytes expressing CD3, CD8, CD19, and HLA-DR, but not CD2, CD4, CD16, and CD25, were significantly lower in FITC-stained versus PE-stained preparations. Correlated analysis of green fluorescence and forward 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 of FITC-anti-CD3-coated lymphocytes complexed with platelet-coated myeloid cells. In studies of the role of lysing agent, essentially no escapees were found in specimens treated with FACS lysing solution, which contains formaldehyde. We therefore included a similar denaturing agent, paraformaldehyde (0.1%), in the AmChl lysing agent, and found that the occurrence of escapees was markedly reduced. These findings show that the escapee phenomenon occurs when using some FITC-conjugated monoclonal antibodies in conjunction with AmChl lysing agent, and can be reduced by inclusion of paraformaldehyde in the lysing agent.