Reduction of escapee formation in flow cytometric analysis of lymphocyte subsets
H E Prince1, J York, D K Kuttner
1American Red Cross Blood Services, Southern California Region, Los Angeles.
Insights
The escape phenomenon in flow cytometry, where cells appear outside gates, is reduced by adding paraformaldehyde to ammonium chloride lysing agents. This method minimizes escapees when using fluorescein isothiocyanate (FITC) conjugates with lymphocyte immunophenotyping.
Area of Science:
- Immunology
- Cell Biology
- Flow Cytometry
Background:
- The escape phenomenon in flow cytometry involves lymphocyte subsets appearing outside their expected scatter gates.
- Limited information exists on factors influencing escapees, including antibody conjugates, fluorochromes, lysing agents, and mitigation strategies.
Purpose of the Study:
- To systematically analyze the escape phenomenon in lymphocyte immunophenotyping.
- To investigate the impact of different antibodies, fluorochromes (FITC vs. PE), and lysing agents on escapee formation.
- To identify methods for reducing the escapee phenomenon.
Main Methods:
- Whole blood specimens were stained with monoclonal antibodies, including FITC and PE conjugates.
- Red blood cells were lysed using ammonium chloride (AmChl) or FACS lysing solution.
- Escapee events were quantified by analyzing forward scatter and green fluorescence, and by dual-color analysis.
Main Results:
- Significantly fewer lymphocytes were gated using FITC conjugates compared to PE conjugates.
- Escapee events, particularly with FITC-anti-CD3, -CD8, and -CD19 antibodies, were observed.
- Dual-color analysis revealed escapees comprised FITC-antibody-coated lymphocytes complexed with platelet-coated myeloid cells.
- Using FACS lysing solution (containing formaldehyde) or AmChl with 0.1% paraformaldehyde markedly reduced escapees.
Conclusions:
- The escape phenomenon is associated with specific FITC-conjugated antibodies and AmChl lysis.
- Escapees can be reduced by including a denaturing agent like paraformaldehyde in the lysing solution.
- These findings provide strategies to improve the accuracy of lymphocyte immunophenotyping by flow cytometry.
Abstract:
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.
