This technical update provides information on the escapee phenomenon, an event that can occur when using monoclonal antibodies for flow cytometric analysis.
Escapees are aggregates of cells (including positively labeled cells) that are excluded from the flow cytometric analysis gate, thus altering the results of the analysis.
The escapee phenomenon can account for the sum of %T + %B + %NK being less than 95% when the lymphocyte gate has been set correctly. This is due to the exclusion of the escapees from the standard lymphocyte gate.
Two dot plots illustrating the escapee phenomenon are shown below. Mononuclear cells from a normal donor were separated from peripheral blood using Ficoll-Paque. The monoclonal antibody used for staining was CD8 FITC from two different manufacturers. The forward light scatter (FSC) vs fluorescence-1 (FL1) dot plots obtained from each manufacturer’s product reveal a population of CD8 + aggregated cells that extend beyond the region of the lymphocyte gate. These events, which show an increased FSC signal, have been identified by sorting as the escapees or aggregated lymphocytes. Since these cells are not included in the standard lymphocyte analysis gate, the result is lower percentage positive values for the antigen of interest.
Figure 1 Region 1 includes the CD8-positive and CD8-negative single lymphocytes within the lymphocyte gate. Region 2 contains the monocyte population. Region 3 includes a population of bright CD8-positive events that are outside of the standard lymphocyte gate.
Use of various monoclonal antibodies such as CD8 and CD4 can cause cell aggregation in immunophenotyping assays, resulting in lower percentage positive values for the antigen of interest. The number of aggregates can vary depending on whether a FITC- or PEconjugated monoclonal antibody is used. When analyzing lymphocyte-gated CD8-positive cells, CD8 FITC conjugates have been shown to yield lower percentage positive values than CD8 PE conjugates for the same donor. Escapees can also be detected when using an unconjugated purified monoclonal antibody and a Goat Anti-Mouse (GAM) Ig FITC second-step reagent.
The number of escapees is partially dependent on the cell preparation method used. A peripheral blood mononuclear cell separation method will result in detection of more escapees than will a whole blood preparation stained and then lysed with FACS Lysing Solution.
- To decrease the number of escapees present, BD recommends using whole blood preparations that have been lysed with FACS Lysing Solution prior to flow cytometric analysis. Refer to the FACS Lysing Solution package insert for a detailed procedure.
- For cell suspensions prepared by Ficoll-Paque separation, it is recommended that a high-protein medium be used, such as phosphate-buffered saline (PBS) containing 2% fetal bovine serum and 0.1% sodium azide.
- Cell suspensions should be vortexed at a low speed, and samples should not be centrifuged with forces greater than 300 x g. Rough handling of cells such as excessive washing or high-speed vortexing can cause increased cell aggregation.
- Avoid the use of agents for monocyte depletion by phagocytosis (iron carbonyl particles) as these can increase the incidence of escapees.
- Even when taking these precautions during sample preparation, cell aggregation can still occur.
For more information, contact the Customer Support Center at your local BD office.
- Jackson A. Basic phenotyping of lymphocytes: selection and testing of reagents and interpretation of data. Clin Immunol Newslett. 1990;10:43-55.